Structure of recombinant human interleukin 5 produced by Chinese hamster ovary cells

The complete peptide map of purified recombinant human interleukin 5 (rhIL-5) was determined to verify its primary structure, glycosylation sites, and disulfide bonding structure. Each peptide fragment generated by Achromobacter protease I (API) digestion was purified and characterized by amino acid analysis and amino acid sequence analysis. After digestion with API, we could identify all the peptides which were expected from human IL-5 cDNA sequence. The analyses of sulfhydryl content in rhIL-5 molecule and disulfide-containing peptide obtained from API digestion indicated that active form of rhIL-5 existed as an antiparallel dimer linked by two pairs of Cys-44 and Cys-86. In addition, we concluded that Thr-3 and Asn-28 were glycosylated. The results indicate that primary structure of rhIL-5 is highly homogeneous and observed heterogeneity is due to the difference in the content of carbohydrate.     

Alteration of receptor/G-protein interaction by putative endogenous protein kinase activity in Dictyostelium discoideum membranes

Membranes of Dictyostelium discoideum cells were incubated under phosphorylation conditions and washed, and the effects on cAMP binding to chemotactic receptors in the absence and presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) were investigated. Most experiments were done with adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), which is a good substrate for many kinases, but the product, protein phosphorothioate, is not easily hydrolyzed by phosphatases. Pretreatment of membranes under phosphorylating conditions with MgATP gamma S alters the site heterogeneity of the cAMP-binding forms, without a significant effect on the total number of binding sites. A similar effect was induced by GTP gamma S under nonphosphorylation conditions. The effects of MgATP gamma S were rapid (t1/2 = 1 min), irreversible, and not induced by Mg2+ or ATP gamma S alone or by magnesium adenylyl imidodiphosphate and magnesium adenylyl (beta, gamma-methylene)diphosphate. MgATP induced a smaller inhibition than MgATP gamma S, which was potentiated by the addition of exogenous cAMP-dependent protein kinase. The effect of MgATP was rapidly reversible; reversibility was reduced by the phosphatase inhibitor NaF. These results suggest that the effects of MgATP gamma S are mediated by an endogenous protein kinase. The major 35S-thiophosphorylated band detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a protein with Mr = 36,000. The phosphorylation of a protein with the molecular weight of the cAMP receptor (Mr = 40,000-45,000) was not observed.     

Effects of IFN on human eosinophils in comparison with other cytokines. A novel class of eosinophil activators with delayed onset of action.

Extracellular killing is regarded as one of the main functions of eosinophils. Therefore, a cytotoxicity assay against antibody-coated Daudi-lymphoma cells was established to measure cytokine effects on peripheral blood eosinophils from healthy volunteers. Optimal time of exposure to cytokines and half optimal concentrations (EC50) were determined and the capability of various cytokines to enhance cytotoxicity of eosinophils was compared. Thus, after 24 h with cytokine, the highest activation of eosinophils was observed with recombinant human rhIFN-gamma (EC50 = 0.2 U/ml), followed by the known activators of eosinophils recombinant human granulocyte/macrophage CSF (rhGM-CSF), rhIL-3, and murine IL-5 (mIL-5). rhIFN-alpha and natural human IFN-beta (nhIFN-beta) enhanced cytotoxicity as well. On the other hand, in short term assays, eosinophils were not stimulated by IFN and the strongest stimulator was rhGM-CSF (EC50 = 0.2 U/ml), followed by rhIL-3, mIL-5, rhTNF, and rhIL-4. With rhTNF-alpha enhancement was more pronounced on freshly isolated eosinophils (EC50 = 0.6 U/ml) and declined with time. No significant stimulation was detected with rhG-CSF, rhIL-1 beta, rhIL-2, rhIL-6, and rhIL-8. On neutrophils, rhIL-8 enhanced cytotoxicity, but the stimulation was weak in relation to other neutrophil activators. Studies on the mechanism of cytotoxic activity revealed that cytotoxicity required opsonization of targets with specific antibody. FMF analysis was performed demonstrating that freshly isolated eosinophils express Fc-gamma RII (CD32), small amounts of Fc-gamma RIII (CD16), but not Fc-gamma RI (CD64). In experiments with blocking antibodies to Fc-gamma R cytotoxicity was restricted to Fc-gamma RII. Expression of Fc-gamma RII was not enhanced by rhGM-CSF, rhTNF-alpha, and mIL-5, but a significant increase in the number of positive cells was observed after incubation with rhIFN-gamma for 24 h (p less than 0.05). In addition, enhanced viability of eosinophils was observed when cultured in the presence of rhIFN-gamma, rhIFN-alpha, rhGM-CSF, and rhTNF-alpha, but not of rhG-CSF and rhIL-2. Thus, IFN appear to be another class of activators of eosinophils, characterized by their delayed type of action.     

Physicochemical characterization of photoaffinity-labeled angiotensin II receptors

Specific receptor sites for angiotensin II (AII) were analyzed in the adrenal cortex and other target tissues including liver, anterior pituitary gland, and smooth muscle, after covalent labeling with the radioactive photoaffinity analog 125I-[Sar1,(4-N3)Phe8]-AII. The photoreactive AII derivative retained high affinity for adrenal receptors and full steroidogenic activity in adrenal glomerulosa cells. In bovine adrenal cortex, covalent labeling of AII receptors by the photoreactive analog was specifically inhibited by [Sar1]AII with an IC50 of about 5 nM. The Mr of the covalent AII-receptor complex during polyacrylamide gel electrophoresis of the labeled protein under reducing conditions was 58,000. Under non-reducing conditions, a minor band with Mr of 105,000 was also observed. Two labeled species were also found during gel permeation chromatography of the detergent-solubilized complex, with Mrs of 64,000 and 86,000. The pl of the solubilized photolabeled complex was absorbed to wheat germ lectin Sepharose 6MB and could be eluted by N-acetylglucosamine. The Mrs of specific AII-binding sites in several target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed significant differences within and between species. The most striking differences were between rat adrenal cortex (79,000) and both rat liver (60,000) and bovine adrenal cortex (58,000). After enzymatic deglycosylation, the Mr of the major component present in the bovine and rat adrenal cortex decreased by 40% and 55% to 35,000 and 34,000, respectively, suggesting that variations in carbohydrate content contribute to the physical heterogeneity of AII receptors in individual target tissues.     

In vivo effects of recombinant human interleukin 6, alone or in combination with local irradiation, on tumor growth in Lewis lung carcinoma-bearing mice

Lewis lung carcinoma (LLC)-bearing mice were used as a mouse model to evaluate effects of recombinant human (rh) interleukin (IL) 6 and local X-irradiation (LR) on the growth of primary tumors and lung metastases. Mice were inoculated s.c. with LLC tumor cells and then treated with rhIL-6 (100 ng/dose) s.c. twice a day (b.i.d.) for 5 days, beginning 6 days after tumor inoculation. LR (800 cGy) was administered to the site of the primary tumor 6 days after tumor inoculation and again 1 wk later. Mice were then observed for survival or sacrificed at day 21 after tumor inoculation to determine size of primary tumor, numbers and size of lung metastases, and other hematological parameters including numbers of granulocyte-macrophage progenitor cells (CFU-gm). The size of the primary tumor and numbers of lung metastases were reduced by rhIL-6. LR enhanced the antitumor effect of rhIL-6 significantly, while LR alone had only a slight antitumor effect. Tumor-associated increases in peripheral blood, femoral marrow, splenic-nucleated cellularity, and marrow and splenic CFU-gm were reduced in mice treated with rhIL-6 plus LR. Prolonged survival time was observed only in tumor-bearing mice treated with rhIL-6 in combination with LR. The antitumor effects in vivo of rhIL-6 appear to be mediated indirectly as rhIL-6 had no effect on proliferation of LLC cells in vitro as assessed by colony and 3H-thymidine incorporation assays. These studies suggest that rhIL-6 may have therapeutic value in the treatment of certain malignancies, especially if used in combination with LR.     

Isolation and characterisation of a human calcitonin-gene-related-peptide receptor

We describe the identification and purification of a receptor for calcitonin-gene-related peptide (CGRP) from human term placenta, using lectin and beta-CGRP-Affigel affinity chromatography. The membrane-bound receptor has an estimated Mr of 240,000, as determined by cross-linking 125I-labelled alpha-CGRP (125I-alpha-CGRP) using discuccinimidyl suberate and SDS/polyacrylamide gel electrophoresis, or of 263,000, as judged by sucrose gradient centrifugation of the soluble partially purified native receptor preparation. Cross-linking studies with disuccinimidyl suberate and N-hydroxysuccinimidyl-4-azidobenzoate using membrane-solubilised, partially purified and CGRP-affinity-purified preparations, show a number of 125I-alpha-CGRP binding subunit(s) of Mr 62,000-68,000. Silver staining of the purified CGRP receptor preparation showed two distinct doublets in this plus a number of minor doublets of lower Mr. The receptor binds human beta-CGRP with greater affinity than alpha-CGRP, and showed little affinity for human calcitonin. Adsorption isotherms and Scatchard analysis of 125I-alpha-CGRP binding to the membrane-bound or soluble purified receptor are consistent, under the conditions used, with a single binding site of high affinity. Molecular cloning at present in progress should define the amino acid sequence and subunit composition of the human placental CGRP receptor, since at present the observed heterogeneity of CGRP-binding proteins may be interpreted in a number of ways, for instance: receptor heterogeneity, variable glycosylation of one of two subunits, or limited proteolysis of a single subunit during purification.     

Effect of photoaffinity labeling on rabbit uterine progesterone receptor

Photoaffinity labeling with [17 alpha-methyl-3H]promegestone ([ 3H]R5020) is an effective technique for the covalent labeling of the progesterone receptor (PR), which allows monitoring of the steroid receptor complex under denaturing conditions. The present study was initiated to evaluate whether photolabeled PR could be used also as a marker for PR under nondenaturing conditions. Accordingly, the effect of irradiation on each component of the reaction was evaluated separately. When [3H]R5020 alone was irradiated, there was a rapid (less than 5 min), light dependent destruction of [3H]R5020, as evident from increased formation of a more polar tritiated product on TLC and a concomitant decrease in the ability of the irradiated preparation to bind to PR. When rabbit uterine PR was irradiated in the absence of steroid, a gradual decrease in the binding capacity was observed, reaching 70% of the nonirradiated control in 10 min. The optimal irradiation time for covalent [3H]R5020-PR complex formation was determined by irradiation for up to 5 min, and separation of the products by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. Specific labeling of proteins of Mr 116,000 and 85,000 was observed, with the rate of labeling of the two being similar, and reaching a plateau by 4 min of irradiation. The photolabeling efficiency ranged from 2 to 12%. Sucrose gradient ultracentrifugation of photolabeled PR revealed that both the irradiated sample and the nonirradiated control sedimented to the same position. Subsequent SDS-polyacrylamide gel electrophoresis of the sucrose gradient peak from the photolabeled sample showed the presence of both labeled proteins of Mr 116,000 and 85,000. In addition, photolabeled rabbit uterine PR (Mr 116,000 and 85,000) could be immunoprecipitated with a guinea pig antiserum raised against rabbit uterine PR. Analysis of the photoaffinity labeling procedure in our system revealed that the photodestruction of [3H]R5020 was very rapid. However, maximal labeling with [3H]R5020 was obtainable with minimal photodestruction of PR which suggests that photolabeled receptor can be used as a marker for PR under nondenaturing conditions.     

Expression, purification and characterization of recombinant human interleukin-2-serum albumin (rhIL-2-HSA) fusion protein in Pichia pastoris

Interleukin-2 (IL-2) plays important roles in variety of immune functions. Recombinant IL-2 has become an important therapeutic protein for therapy of melanoma and renal cell carcinoma. Previously, it was proved that the therapeutic efficacy of rIL-2 expressed in Saccharomyces cerevisiae was improved by prolonging its in vivo half-life through genetic fusion with albumin. In this study, a fusion protein composed of hIL-2 genetically fused to HSA was expressed as a secretory protein under AOX1 (alcohol oxidase 1) promoter in Pichia pastoris. An effective strategy was established to express rhIL-2-HSA fusion protein in 5L scale and the optimal purification procedure was investigated. The purity of rhIL-2-HSA in final product was about 95%. The purified rhIL-2-HSA fusion protein could be recognized by both anti-hIL-2 and anti-human serum albumin monoclonal antibody. Bioactivity analysis showed that the purified rhIL-2-HSA fusion protein displayed high level activity on proliferation in IL-2 dependent manner in CTLL2-cells. rhIL-2-HSA fusion protein also showed a extended half-life in plasma compared with IL-2 when tested in a BALB/c mouse model. This study provides an alternative strategy for large-scale production of bioactive rhIL-2-HSA fusion protein using P. pastoris as an expression host.     

A potential diagnostic reagent for bovine cysticercosis

A fraction of larval Taenia hydatigena cyst fluid was shown to have high sensitivity and specificity in the enzyme-linked immunosorbent assay (ELISA) for the detection of bovine antibodies to the heterologous parasite Taenia saginata. This antigenically active lipoprotein fraction was isolated by ultracentrifugal density flotation using either ammonium sulfate (specific gravity = 1.231 g per ml) or NaCl/KBr (specific gravity = 1.225 g per ml), followed by ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that this fraction was composed of high molecular weight (65,000 to 77,000 Mr) and low molecular weight (9,500 to 16,000 Mr) proteins. Electrophoresis under non-denaturing conditions in either acrylamide (5%) or agarose (1%) resulted in 1 major diffuse band staining for both protein and lipid. The high and low molecular weight proteins observed on SDS-PAGE under reducing conditions could not be resolved by gel filtration chromatography and emerged as a single lipoprotein peak. This T. hydatigena cyst fluid fraction appears promising as a diagnostic reagent in the ELISA for bovine cysticercosis.     

Changes in exposed membrane proteins during in vitro capacitation of boar sperm

Exposed plasma membrane proteins were labeled with 125I before and after incubation of boar sperm under capacitating conditions. Labeled protein profiles were compared to the ability of the sperm to penetrate zona-free hamster ova. Quantitatively, the labeled sperm membrane proteins were primarily low Mr prior to capacitation. The majority of the labeled seminal plasma protein was also low Mr. After capacitation, two new proteins (64,000 Mr and 78,000 Mr) were labeled. Sperm did not exhibit these exposed membrane proteins when incubated under noncapacitating conditions. Appearance of these proteins was not correlated to the percentage of acrosome-reacted sperm. Although the 64,000 Mr protein was not consistently observed, the relative labeling of the 78,000 Mr protein was highly correlated with the ability of sperm to fuse with zona-free hamster ova. The 78,000 Mr protein may be a sperm protein involved in fusion with the egg plasma membrane.     

Human high molecular weight kininogen as a thiol proteinase inhibitor: presence of the entire inhibition capacity in the native form of heavy chain

High molecular weight (HMW) kininogen was purified from fresh human plasma by two successive column chromatographies on DEAE-Sephadex A-50 and Zn-chelate Sepharose 4B. The purified HMW kininogen appeared to be a single band on sodium dodecyl sulfate (SDS)-polyacrylamide disc gel electrophoresis in both the presence and absence of beta-mercaptoethanol. However, it gave two bands on nonreduced SDS-polyacrylamide slab gel electrophoresis, a major band of dimeric form (Mr 200 000, ca. 95%) and a minor band of monomeric form (Mr 105 000, ca. 5%). Under reduced conditions, the dimeric form was converted stoichiometrically to a monomeric form (Mr 110 000), and the monomeric form observed under nonreduced conditions (Mr 105 000) was converted to a heavy chain (Mr 60 000) and a light chain (Mr 50 000). The formation of a dimer of HMW kininogen was also confirmed by an immunoblotting experiment. This unique property of intact HMW kininogen to form a dimer was further utilized in studies on the kininogens and their derivatives as thiol proteinase inhibitors. The purified HMW kininogen strongly inhibited the caseinolytic activities of calpain I, calpain II, and papain but not those of trypsin, chymotrypsin, and thermolysin, indicating that it was a group-specific inhibitor for thiol proteinases. When HMW kininogen was reduced with 0.14 or 1.4 M beta-mercaptoethanol, its inhibitory activity was partially or mostly inactivated, but on subsequent air oxidation its activity was almost completely recovered. In addition, kinin-free and fragment 1,2 free HMW kininogen showed higher inhibitory activity than the intact HMW kininogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of hematologic and immunologic effects of high dose chemotherapy by interleukin-2 in a murine tumor model.

To determine if intensive chemotherapy consisting of cyclophosphamide (C), etoposide (E), and cisplatin (P) (CEP) may be usefully combined with recombinant human interleukin-2 (rhIL-2), we examined a murine tumor model designed to approximate a common clinical situation: macroscopic, drug-resistant cancer. Using C57BL/6 mice with extensive tumor burden 10 days after intravenous B16 melanoma cell injection, we observed (1) C, E, and P synergize to enhance survival but do not cure mice at the highest tolerable dose (C = 200 mg/kg, E = 60 mg/kg, and P = 3 mg/kg); (2) rhIL-2 at 3 x 10(5) U (subcutaneously) daily for 4 days administered 10-18 days after B16 injection significantly improves survival; (3) CEP plus rhIL-2 is more effective than CEP alone only when rhIL-2 is administered before CEP; (4) CEP suppresses IL-2-induced lymphokine-activated killer cell activity in the spleen; and (5) rhIL-2 protects mice incompletely from the immunologic and hematologic suppression of CEP. Our results suggest that intensive chemotherapy combined with rhIL-2 may be beneficial. The success of any such combination may be schedule dependent.     

Identification of surfactant proteolipid SP-B in human surfactant and fetal lung

Surfactant proteolipid (SP-B) is one of several hydrophobic peptides detected in organic extracts of pulmonary surfactant and associated with the dramatic surface-active properties of surfactant phospholipids. In the present study human SP-B was identified as a protein with a relative molecular weight (Mr) of 7,500-8,000 under reducing conditions; protein of Mr 18,000 was detected under nonreducing conditions by immunoblot analysis of organic extracts of bovine and human surfactant utilizing an antiserum directed against a 60-amino acid synthetic SP-B peptide. This peptide antiserum was subsequently used to identify SP-B in explant cultures of 18- to 23-wk gestation human fetal lung. Immunoprecipitation of explants labeled with [35S]methionine after 48 h of culture identified proteins of Mr 40,000-42,000, 25,000, and 18,000 after electrophoresis under nonreducing conditions. The Mr 18,000 form was reduced to Mr 7,500-8,000 in the presence of beta-mercaptoethanol. These molecular forms likely represent the SP-B precursor protein, a proteolytic intermediate, and the mature SP-B peptide, respectively. Immunocytochemistry with the peptide antiserum localized SPL(Phe) in granular inclusions in the apical region of type II-like epithelial cells, a pattern of staining similar to that observed for the major surfactant-associated protein of Mr 26,000-38,000 (SP-A). SP-B is a novel pulmonary surfactant-associated protein that is synthesized by the human alveolar type II epithelial cell as an Mr 40,000-42,000 precursor that is subsequently proteolytically processed to Mr 7,500-8,000.     

Stoichiometry of the interaction of human plasma alpha 1-proteinase inhibitor with subtilisin BPN'

Interaction of human plasma alpha 1-proteinase inhibitor (alpha 1PI) with subtilisin BPN' was assessed by spectrophotometric determination of the inhibitory capacity and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). During the course of incubation of the enzyme and the inhibitor (E : I = 1 : 7.5) at pH 8.0 about 17% of the enzyme activity which had been inhibited initially was regenerated, indicating a temporary type of inhibition. The results of the titration experiments indicate that 9.8 mol of the inhibitor is required to inhibit 1 mol of the enzyme completely. However, patterns of 5% disc SDS-PAGE under non-reducing conditions revealed only an equimolar complex (Mr80K) of alpha 1PI with the enzyme and no other higher Mr component than the native inhibitor (Mr 56K). On the other hand, complete dissociation of the complex occurred under reducing conditions, producing an enzymatically modified inhibitor. When 5 21% gradient slab SDS-PAGE was employed, no complex formation was observed under either reducing or non-reducing conditions. With the gradient gel system, dissociation of the equimolar complex produced different forms of the inhibitor, that is, regeneration of an intact alpha 1PI under non-reducing conditions and an enzymatically modified form under reducing conditions. All these results indicate that the complex formed between subtilisin BPN' and human alpha 1PI is not so stable as that of the inhibitor with bovine chymotrypsin and that no covalent bond may be involved in the complex formation. The results also indicate that human alpha 1PI is not an effective inhibitor of subtilisin BPN' and behaves like a substrate for the enzyme.     

Biosynthesis of chick type VI collagen. I. Intracellular assembly and molecular structure

A monospecific rabbit antiserum to pepsin-extracted chick gizzard type VI collagen was used to characterize the intact forms of type VI collagen in tissues and cultured cells. Immunoblotting of gizzard extracts revealed polypeptides of Mr ranging from 260,000 to 140,000. Components of about Mr = 260,000, 150,000, and 140,000, each with a different peptide profile, were immunoprecipitated from labeled matrix-free chick embryo cells. Cleavage of the immunoprecipitated polypeptides with pepsin generated pepsin-resistant fragments of about Mr = 70,000, 55,000, and 45,000 that represent the alpha 1(VI), alpha 2(VI), and alpha 3 (VI) fragments. Immunoblotting with affinity-purified antibodies indicated that the Mr = 150,000 is the intact parent polypeptide of the alpha 1(VI) pepsin; the Mr = 140,000 of the alpha 2(VI) pepsin, and the Mr = 260,000 of the alpha 3(VI) pepsin. Association of the three parent chains was studied by pulse-chase experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis under nonreduced conditions. A complex of Mr = 500,000 is already present intracellularly at the end of a 7-min pulse and increases considerably with time while the three unassembled chains show a comparable decrease. After 5-15 min of chase larger forms appeared along with small amounts of aggregated material that did not enter the gel. Analysis of the immunoprecipitate by diagonal electrophoresis indicated that the component of Mr = 500,000 and the larger forms dissociated into the Mr = 260,000, 150,000, and 140,000 polypeptides. Sedimentation profile of a labeled cell extract on a 5-20% sucrose gradient under nondenaturing conditions confirmed the presence of three different peptides in the complex.     

Local interleukin-2 therapy in bovine ocular squamous cell carcinoma

Summary Five cows bearing bovine ocular squamous cell carcinoma (BOSCC) were treated with low doses of recombinant human interleukin-2 (rhIL-2). A dose of 2500 U rhIL-2 was injected intralesionally and another 2500 U were injected into the subparotid regional lymph node once a day during a period of 5 consecutive days. This cycle of 5 days was repeated after an interval of 2 days. Total regression of the tumor was observed in three out of five animals. One cow showed tumor regression (> 80%) accompanied by metastases to the regional lymph node that were observed from the fifth week after the beginning of the treatment. Growth of the tumor of the fifth animal was retarded after treatment. In vitro proliferation of peripheral blood lymphocytes was investigated in two animals and tumor-infiltrating lymphocytes in one animal during incubation in various rhIL-2 concentrations. Cytotoxic activity of both cell populations against P815, Yac-1 and BOSCC-derived cell lines increased during incubation with rhIL-2. Cultured BOSCC-infiltrating lymphocytes showed predominant killing of the BOSCC-derived autologous cell line after 4 weeks of culture. Preliminary phenotype analysis did not give conclusive results with respect to the types of cells responsible for killing.     

Cell-free synthesis and characterization of human adrenocortical pro-adrenodoxin

Poly(A+)-RNAs were extracted from human hyperplasic adrenocortical tissue and translated in a wheat germ cell-free system in the presence of [35S]-methionine. Labeled immuno-reactive adrenodoxin (ADX)-like material was immunoisolated and examined following mono and bi-dimensional electrophoretic analysis. Bovine mRNA translation products were analysed under similar conditions. While it was confirmed that bovine ADX was synthesized as a precursor of Mr 21 kDa, human pro-ADX was characterized for the first time as a somewhat larger moiety (24 kDa). On the other hand, both human and bovine mature mitochondrial ADX showed a Mr of 12 kDa. Electrophoretic study disclosed that the human, as well as the bovine pro-ADX could be resolved into several components differing by their pHi (6.5 and 6.9 for h-proADX and 5.9, 6.1 and 6.2 for b-proADX, respectively). This molecular heterogeneity might be explained by discrete disparity in the pro-adrenodoxin amino acid contents.     

Pig MAP/ITIH4 and haptoglobin are interleukin-6-dependent acute-phase plasma proteins in porcine primary cultured hepatocytes.

The acute-phase expression of pig MAP (major acute-phase protein)/ITIH4 (inter-alpha-trypsin inhibitor heavy chain 4) and haptoglobin were analysed in primary cultures of isolated pig hepatocytes in response to recombinant human (rh) cytokines: tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6), as well as to bacterial lipopolysaccharide (LPS). Analysis of pig MAP/ITIH4 and haptoglobin mRNAs was carried out by RT-PCR amplification. Secreted proteins from the cytokine-treated hepatocytes were quantified by immunochemical techniques. Time-course and dose-response experiments show that pig MAP/ITIH4 and haptoglobin belong to the type II acute-phase proteins, as they are specifically induced by rhIL-6 and not by rhTNF-alpha or rhIL-1. Stimulation of cultured pig hepatocytes with rhIL-6 for 48 h at doses of 1000 U.mL-1 showed a fourfold to fivefold increase in pig MAP/ITIH4 concentration in the medium, while the concentration of haptoglobin only increased twofold. A similar increase in the concentration of pig MAP/ITIH4 was also observed in media of LPS-treated hepatocytes with the simultaneous generation of IL-6 by the Kupffer cells present in the cultures. Albumin secretion decreased after stimulation with doses of 100 or 1000 U.mL-1 rhTNF-alpha, rhIL-1 or rhIL-6. Therefore, it can be concluded that pig MAP/ITIH4 behaves as a major acute-phase protein produced by porcine hepatocytes under the effect of inflammatory cytokines.     

Human A673 cells secrete high molecular weight EGF-receptor binding growth factors that appear to be immunologically unrelated to EGF or TGF-alpha

Extracts of serum-free conditioned medium from human rhabdomyosarcoma A673 cells contain high molecular weight (HMW) transforming growth factors (TGFs) that can be partially purified by Bio-Gel P-100 and carboxymethyl (CM)-cellulose chromatography (Todaro et al: Proc Natl Acad Sci USA 77:5258, 1980). Reverse-phase high performance liquid chromatography (HPLC) revealed a principal peak of epidermal growth factor (EGF) radioreceptor assay (RRA) activity and anchorage-independent growth (AIG) activity that coeluted with 25-26% acetonitrile. If a trailing shoulder of EGF RRA activity from the CM-C chromatography was included in the material for HPLC analysis, additional active fractions were observed at 21-22% acetonitrile. Importantly, both active regions from HPLC failed to compete in radioimmunoassays under reduced and denatured conditions for human EGF (hEGF), human TGF-alpha (hTGF-alpha), or rat TGF-alpha (rTGF-alpha) and failed to give positive signals in Western blots under conditions in which TGF-alpha was readily detected when using an antisera raised against the 17 C-terminal amino acids of rTGF-alpha. Nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed EGF RRA and AIG activities in gel slices corresponding to Mr 15,000 and 22,000 in the 25-26% acetonitrile eluate and Mr 15,000, 20,000, 27,000, and 48,000 in the 21-22% acetonitrile eluate. The presence of multiple forms of EGF-receptor-binding peptides produced in vitro suggest size heterogeneity and possible immunologic diversity among high molecular weight members of the EGF/TGF-alpha family of growth-promoting polypeptides.