[Factor analysis of effective platelet-producing ability of fetal liver-derived cells].

Objective: To study the different factors affecting platelet production post transplantation of hematopoietic stem cells (HSCs) isolated from different sources in order to explore novel options for treating platelet depletion following HSCs transplantation. Methods: HSCs and their downstream derivatives including myeloid and lymphoid cells (i.e., collective of mononuclear cells (MNCs)), were isolated from E14.5 fetal liver (FL) and bone marrow (BM) of 8-week-old mice by Ficoll separation technique. These cells were subsequently transplanted into the tibia bone marrow cavity of recipient mice post lethal myeloablative treatment in order to construct the FL-MNCs and BM-MNCs transplantation mouse model. Routine blood indices were examined in these recipient mice. The chimeric rate of donor cells in recipient peripheral blood cells were determined by flow cytometry. Different groups of cells involved in platelet reconstruction were analyzed. CD41+megakaryocytes were sorted from fetal liver or bone marrow using magnetic beads, which were then induced to differentiate into platelets in an in vitro assay. Quantitative RT-PCR was used to detect the expression of platelet-related genes in CD41+megakaryocytes from the two sources. Results: Both the FL-MNCs and the BM-MNCs transplantation groups resumed normal hematopoiesis at the 4th week after transplantation, and the blood cells of the recipient mice were largely replaced by the donor cells. Compared with the mice transplanted with BM-MNCs, the platelet level of mice transplanted with FL-MNCs recovered faster and were maintained at a higher level. At week 4, the PLT level of the FL-MNCs group was (1.45±0.37)×1012/L, and of the BM-MNCs group was (1.22±0.24)×1012/L, P<0.05. The FL-MNCs contain a higher proportion of hematopoietic stem cells (Lin-Sca-1+c-Kit+)(7.60%±1.40%) compared to the BM-MNCs (1.10%±0.46%), P<0.01; the proportion of the megakaryocyte progenitor cells (Lin-Sca-1-c-Kit+CD41+CD150+) and mature megakaryocyte cells (CD41+CD42b+), also differ significantly between the FL-MNCs (3.05%±0.22%, 1.60%±0.06%, respectively) and the BM-MNCs (0.15%±0.02%, 0.87%±0.11%, respectively) groups, both P<0.01. In vitro functional studies showed that FL-MNCs-CD41+megakaryocytes could produce proplatelet-like cells more quickly after induction, with proplatelet-like cells formation on day 3 and significant platelet-like particle formation on day 5, in contrast to bone marrow-derived BM-MNCs-CD41+megakaryocytes that failed to form proplatelet-like cell on day 5. In addition, FL-MNCs-CD41+cells expressed higher levels of platelet-related genes, Mpl (3.25-fold), Fog1 (3-fold), and Gata1 (1.5-fold) (P<0.05). Conclusion: Compared with the BM-MNCs group, the FL-MNCs transplantation group appears to have a more efficient platelet implantation effect in the HSCs transplantation recipient in vivo, as well as a higher platelet differentiation rate in vitro. This might be related to a higher proportion of megakaryocytes and higher expression levels of genes such as Mpl, Fog1, and Gata1 that could be important for platelet formation in FL-MNCs-CD41+cells. Further exploration of the specific functions of these genes and the characteristics of the different proportions of the donor cells will provide valuable clues for the future treatment of platelets reconstitution after HSCs transplantation clinically.

[The effect of DNA hydroxymethylase Tet2 on γ globin activation in the treatment of ß-thalassemia].

Objective: To study the function of ten-eleven translocation 2 (Tet2) in γ globin gene expression in patients with ß- thalassemia. Methods: Gamma globin expression was induced by 5-azacytidine and Tet2 gene expression was knocked down by short hairpin RNA (shRNA) in a human immortalized myelogenous leukemia K562 cell line. The global 5-hydroxymethylcytosine (5hmC) level was measured by an ELISA kit. 5hmC level of γ globin gene was quantified by sulfite sequencing. The mRNA level of Tet2, γ globin, and related transcription factors Nfe4 and Klf1 were quantified by real-time PCR. Results: Tet2 knockdown resulted in a decreased global 5hmC level from 0.14% to 0.03% as of the control group in K562 cells. The expression of γ globin was enhanced after 5-azacytidine treatment in vitro. However, γ globin mRNA level in Tet2 knockdown cells was only 55% as that in control group. The CG sites on γ globin gene were unmethylated. As Tet2 was down-regulated, the expression levels of Nfe4 and Klf1 decreased by about 80% and increased to 3.5 folds, respectively. Conclusions: Tet2 appears to maintain 5hmC level and facilitates γ globin gene activation. Moreover, Tet2 more likely regulates γ globin expression via affecting transcription factors rather than the gene itself. Thus, Tet2 could be a potential therapeutic target for ß thalassemias.

Integrated nuclear fluorescence and expression of hormone-binding sites in malignant pleural effusions.

OBJECTIVE: To investigate potential disease- and prognosis-associated nuclear and cellular features from cell properties in a prospective study on malignant pleural effusions. STUDY DESIGN: Integrated nuclear fluorescence and the expression of binding capacities of carrier-immobilized estradiol, progesterone and testosterone; and of labeled sarcolectin; and the presence of calcyclin were measured in 50 cases with proven malignant pleural effusions (10 mesotheliomas, 40 metastasizing tumors). A double fluorescence technique using the fluorochrome DAPI and a Texas Red-based avidin-biotin detection system were applied. Detailed clinical data, including the follow-up for up to 40 months, were included. RESULTS: Pleural effusions in all patients with mesotheliomas occurred prior to (9/10) or at the time of histologic confirmation. Mesotheliomas had the highest tumor cell fraction (12.4%) in S phase and breast carcinomas the lowest (10.7%). More than 80% of malignant cells expressed binding capacities for the applied probes. A statistically significant correlation was noted between the S-phase-related tumor cell fraction and the expression of progesterone receptors. Survival was associated with tumor origin, treatment by pleurodesis, and certain cytometric and histochemical features. CONCLUSION: The immunofluorescence double-staining technique can be applied successfully in malignant effusions to combine DNA measurements with those of immunohistochemical and ligand histochemical reactivity.

Correlation of galectin-3/galectin-3-binding sites with low differentiation status in head and neck squamous cell carcinomas.

The accurate determination of levels of differentiation is of prognostic value in human head and neck squamous cell carcinomas (HNSCCs). Because the deliberate selection of biochemical determinants accompanying certain stages of differentiation can refine the predictive power of histochemical assessments, the application of the quantitative evaluation of staining distribution and intensity by computer-assisted microscopy is one prerequisite to potential improvements. We used 2 innovative approaches with peanut agglutinin based on encouraging results with respect to common lectin-histochemistry. First, we used a custom-made neoglycoprotein to monitor the presence of Thomsen-Friedenreich (T) antigen-binding sites. Second, we measured the presence of 2 galectins immunohistochemically and, at the same time, measured lectin-histochemically the presence of accessible ligands for the endogenous lectins. We also monitored the presence of calcyclin, a protein with relevance to cell cycle progression or exocytosis. With 61 cases of HNSCC as their basis, including 31 oral, 20 laryngeal, and 10 hypopharyngeal lesions, the data show that the main modifications observed in connection with a loss of differentiation are related to a modification in the levels of both galectin-3/galectin-3-binding site and T-antigen/T-antigen-binding site expressions. The data obtained also suggest that galectin-3 could act as an acceptor site for the T antigen. Because the level of differentiation is known to be indicative of the recurrence rate in HNSCCs and our data clearly indicate that galectin-3 and the T antigen (and their respective binding sites) are involved in dedifferentiation processes, further investigation is warranted into the roles of galectins in HNSCC tumor progression and recurrence analysis.

Conserved extracellular cysteine pair in the M3 muscarinic acetylcholine receptor is essential for proper receptor cell surface localization but not for G protein coupling.

Most G protein-coupled receptors contain a conserved pair of extracellular cysteine residues that are predicted to form a disulfide bond linking the first and second extracellular loops. Previous studies have shown that this disulfide bond may be critical for ligand binding, receptor activation, and/or proper receptor folding. However, the potential importance of the two conserved cysteine residues for proper receptor cell surface localization has not been investigated systematically. To address this issue, we used the rat M3 muscarinic receptor as a model system. Most studies were carried out with a modified version of this receptor subtype (lacking potential N-glycosylation sites and the central portion of the third intracellular loop) that could be readily detected via western blot analysis. Cys-->Ala mutant receptors were generated, transiently expressed in COS-7 cells, and then examined for their subcellular distribution and functional properties. ELISA and immunofluorescence studies showed that the presence of both conserved cysteine residues (corresponding to C140 and C220 in the rat M3 muscarinic receptor sequence) is required for efficient expression of the M3 muscarinic receptor on the cell surface. On the other hand, these residues were found not to be essential for protein stability (determined via immunoblotting) and receptor-mediated G protein activation (studied in second messenger assays). These results shed new light on the functional role of the two extracellular cysteine residues present in most G protein-coupled receptors.

Identification and molecular characterization of m3 muscarinic receptor dimers.

Several studies suggest, but do not prove directly, that muscarinic receptors may be able to form dimeric or oligomeric arrays. To address this issue in a more direct fashion, we designed a series of biochemical experiments using a modified version of the rat m3 muscarinic receptor (referred to as m3') as a model system. When membrane lysates prepared from m3' receptor-expressing COS-7 cells were subjected to Western blot analysis under non-reducing conditions, several immunoreactive species were observed corresponding in size to putative receptor monomers, dimers, and oligomers. However, under reducing conditions, the monomeric receptor species represented the only detectable immunoreactive protein, consistent with the presence of disulfide-linked m3 receptor complexes. Similar results were obtained when native m3 muscarinic receptors present in rat brain membranes were analyzed. Control experiments carried out in the presence of high concentrations of the SH group alkylating agent, N-ethylmaleimide, suggested that disulfide bond formation did not occur artifactually during the preparation of cell lysates. The formation of m3' receptor dimers/multimers was confirmed in coimmunoprecipitation studies using differentially epitope-tagged m3' receptor constructs. In addition, these studies showed that m3' receptors were also able to form non-covalently associated receptor dimers and that m3' receptor dimer formation was receptor subtype-specific. Immunological studies also demonstrated that m3' receptor dimers/multimers were abundantly expressed on the cell surface. Site-directed mutagenesis studies indicated that two conserved extracellular Cys residues (Cys-140 and Cys-220) play key roles in the formation of disulfide-linked m3' receptor dimers. These results provide the first direct evidence for the existence of muscarinic receptor dimers and highlight the specificity and molecular diversity of G protein-coupled receptor dimerization/oligomerization.

Structure-function analysis of muscarinic receptors and their associated G proteins.

Each member of the muscarinic receptor family (M1-M5) can interact only with a limited subset of the many structurally closely related heterotrimeric G proteins expressed within a cell. To understand how this selectivity is achieved at a molecular level, we have used the G(i/0)-coupled M2 and the Gq/11-coupled M3 muscarinic receptors as model systems. We developed a genetic strategy involving the coexpression of wild type or mutant muscarinic receptors with hybrid or mutant G protein alpha subunits to identify specific, functionally relevant receptor/G protein contact sites. This approach led to the identification of N- and C-terminal amino acids on alpha(q) and alpha(i) that are critical for maintaining proper receptor/G protein coupling. Moreover, several receptor sites were identified that are likely to be contacted by these functionally critical G alpha residues. To gain deeper insight into muscarinic receptor structure, we recently developed a cysteine disulfide cross-linking strategy, using the M3 muscarinic receptor as a model system. Among other structural modifications, this approach involves the removal of most native cysteine residues by site-directed mutagenesis, the insertion of three factor Xa cleavage sites into the third intracellular loop, and systematic 'reintroduction' of pairs of cysteine residues. Following treatment of receptor-containing membrane preparations with factor Xa and oxidizing agents, disulfide cross-linked products can be identified by immunoprecipitation and immunoblotting studies. This approach should greatly advance our knowledge of the molecular architecture of muscarinic and other G protein-coupled receptors.

Structure-function analysis of muscarinic acetylcholine receptors.

The structural basis underlying the G protein coupling selectivity of different muscarinic receptor subtypes was analyzed by using a combined molecular genetic/biochemical approach. These studies led to the identification of key residues on the receptors as well as the associated G proteins that are critically involved in determining proper receptor/G protein recognition. Mutational analysis of the m3 muscarinic receptor showed that most native cysteine residues are not required for productive receptor/G protein coupling. The putative extracellular disulfide bond was found to be essential for efficient trafficking of the receptor protein to the cell surface but not for receptor-mediated G protein activation.

Association of prognosis in surgically treated lung cancer patients with cytometric, histometric and ligand histochemical properties: with an emphasis on structural entropy.

OBJECTIVE: To explore new tumor features for refined category formation that permits the tailoring of individualized treatment schemes in lung cancer. STUDY DESIGN: Survival data on patients from six independent studies on cases with surgically treated lung cancer, primary lung carcinoids or metastasizing breast carcinoma (including data on primary breast carcinoma) were analyzed by nonhierarchic multivariant discriminant analysis with respect to a set of cytometric/histometric and immunohistochemical/ligand histochemical parameters. The number of stem lines, S-phase-related tumor cell fraction and the extent of structural entropy and its current were measured. In addition, the expression of binding capacities for histo-blood group trisacharides, galectins, the alpha/beta-interferon antagonist sarcolectin, the lymphokine macrophage migration inhibitory factor and a monoclonal antibody to the Le(y) epitope was monitored for insight into aspects of immunologic and biologic behavior. RESULTS: In all studies, a correlation between tumor parameters, according to TNM stage and survival, was seen. In order to refine this category formation, at least certain selected features should provide an even more stringent association than TNM stages. Indeed, statistical correlation of the cytometric and histometric parameters as well as the expression of receptors for the two histo-blood group trisaccharides, ligands for the galectins (CL-16, CL-14) and macrophage migration inhibitory factor was stronger than that of TNM stage. A large amount of the current of structural entropy was especially highly significantly associated with poor survival. This observation could be verified in each of the different studies. CONCLUSION: The obtained data strongly support the notion that thermodynamic evaluation of tumor growth focusing on the "entropy distance" of the tumor from its environment is a promising perspective warranting extended studies. Additionally, glycohistochemical features, including binding capacities for histo-blood group trisaccharides, have the potential to aid in establishment of a biologic marker set for tumor staging.

Preneoplasia-associated expression of calcyclin and of binding sites for synthetic blood group A/H trisaccharide--exposing neoglycoconjugates in human lung.

Development of preneoplastic lesions in human lung is supposed to be accompanied with alterations of distinct biochemical features which might functionally be crucial for this alteration. To contribute to the definition of such determinants in peripheral lung parenchyma, the files of the Department of Pathology, Thoraxklinik, (a total of 2890 cases) were screened for respective tissue specimens. Seventy one cases with complete clinical documentation were found and an age-, sex-, and disease-matched control group was formed. When compared to control group patients, especially the tumor free cases with preneoplastic aberrations revealed a history of exposure to external noxes. Several probes with assumed relevance were tested with the panel of specimens for both groups, focussing on comparative analysis of alveolar lining cells. In addition to labelled neoglycoconjugates which include tissue lectin-seeking probes that expose mono-, di- and blood group-related trisaccharides, presence of calcium- and annexin-binding calcyclin, of complement component C5b, of the lymphokine macrophage migration inhibitory factor, and of ligands of the serum amyloid P component was evaluated. Compared to normal cells at the alveolar surface in controls, the preneoplastic cells displayed an apparent down-regulation of expression of A/H-trisaccharide-specific binding sites and an upregulation of expression of calcyclin. These three characteristics correlated with the phenotypic alterations and encourage further studies to elucidate the functional significance of reduced expression of the glycoligand-specific sites and the presence of this member of the S100-family of Ca(2+)-binding proteins.

Fatty acylation of the rat and human asialoglycoprotein receptors. A conserved cytoplasmic cysteine residue is acylated in all receptor subunits.

Functional rat or human asialoglycoprotein receptors (ASGP-Rs) are hetero-oligomeric integral membrane glycoproteins. Rat ASGP-R contains three subunits, designated rat hepatic lectins (RHL) 1, 2, and 3; human ASGP-R contains two subunits, HHL1 and HHL2. Both receptors are covalently modified by fatty acylation (Zeng, F.-Y., Kaphalia, B. S., Ansari, G. A. S., and Weigel, P. H. (1995) J. Biol. Chem. 270, 21382-21387; Zeng, F.-Y., Oka, J. A., and Weigel, P. H. (1996) Biochem. Biophys. Res. Commun. 218, 325-330). We report here that the single Cys residue in the cytoplasmic domain of each RHL or HHL subunit is fatty acylated. The degree of acylation is >/=90% per subunit. Deacylation of affinity-purified ASGP-Rs with hydroxylamine results in the spontaneous formation of dimers through reversible disulfide bonds, indicating that deacylation concomitantly generates free thiol groups. Reaction of hydroxylamine-treated ASGP-R with [14C]iodoacetamide resulted in the specific incorporation of radioactivity into all RHL and HHL subunits, verifying that fatty acids are attached via thioester linkages. To identify the Cys residue involved in the thioester linkages, 14C-carboxyamidomethylated RHL subunits were separated by SDS-polyacrylamide gel electrophoresis and digested in-gel with trypsin, and the resulting peptides were separated by reverse-phase high performance liquid chromatography. Amino acid sequence of radioactive peptides revealed that Cys35 in RHL1 and Cys54 in RHL2 and RHL3 were radiolabeled and, therefore, are fatty acylation sites. Fatty acylation of HHL subunits was analyzed by site-directed mutagenesis. Metabolic labeling of Cos7 cells transfected with wild type HHL1 cDNA resulted in substantial incorporation of [3H]palmitate into purified HHL1. Incorporation of [3H]palmitate into a C36S mutant of HHL1 was negligible ( approximately 1%) compared with wild type. This result also shows that Cys57 within the transmembrane domain of HHL1 is not normally palmitoylated. We conclude that Cys35 in RHL1, Cys54 in RHL2 and RHL3, and Cys36 in HHL1 are fatty acylated. Cys57 in HHL1 and probably Cys56 in RHL1 are not palmitoylated.

The human asialoglycoprotein receptor is palmitoylated and fatty deacylation causes inactivation of state 2 receptors.

We report here for the first time that ASGP-Rs expressed in the human hepatoma cell lines HuH-7 and HepG2 are fatty acylated. Cells were metabolically labeled with [3H]palmitate and active ASGP-Rs, which contain two subunits (HHL1 and HHL2), were purified by affinity chromatography and subjected to nonreducing SDS-PAGE and fluorography. [3H]Palmitate was covalently incorporated into both HHL1 and HHL2. When gel slices containing HHL1/HHL2 were treated at neutral pH with 1 M hydroxylamine, but not 1 M Tris, > 95% of the radioactivity was removed, indicating that the attachment of palmitate to ASGP-Rs is to cysteines. Furthermore, the same mild hydroxylamine treatment caused partial ASGP-R inactivation; 50-70% of receptors corresponding to the previously characterized State 2 ASGP-Rs were inactivated. We conclude that both HHL1 and HHL2 are covalently modified by fatty acylation, which may regulate the ligand-binding activity of human State 2 ASGP-Rs. We propose that fatty acylation/deacylation of cytoplasmic domains is a general mechanism by which extracellular ligand-binding activity of oligomeric transmembrane receptors can be regulated.

Differential expression of calcyclin and its accessible ligands in various types of cutaneous tumors.

Calcyclin is the product of a gene that is regulated in dependence of the cell cycle in fibroblasts in vitro. It has recently been proven to be a sialic acid-binding protein in vitro and in the case of mammalian tissues to bind specifically to annexin II, annexin VI, annexin XI, and glyceraldehyde-3-phosphate dehydrogenase in a Ca(2+)-dependent manner. Since calcyclin can be labelled without impairment of its binding activity, it can be employed as a histochemical tool to localize its accessible ligands. Concomitantly, immunohistochemical localization of calcyclin with a specific antibody is warranted. By using histochemical and immunohistochemical techniques, the expression of calcyclin and its accessible binding sites are demonstrated in serial sections of normal skin and benign, pre-cancerous and malignant tumors of the skin, namely in verruca vulgaris, papillary hidradenoma, syringoma, keratoacanthoma, Bowen's disease, squamous cell carcinoma, melanocytic naevi, primary and metastatic malignant melanoma and non-Hodgkin lymphoma (NHL) of the skin. Cytoplasmic and nuclear expression of calcyclin and its ligands is unexceptionally found in normal skin, epithelial tumors and benign melanocytic tumors. Presence of calcyclin and calcyclin-binding sites is detected in more than 80% of tumor cells in the epithelial lesions. In the group of melanomas and lymphomas heterogeneity is obvious. The application of annexin-specific antibodies raises evidence that members of this protein family co-localize with calcyclin in situ to some extent. These findings suggest that calcyclin and accessible calcyclin-binding molecules, like certain annexins, may be differentially regulated in melanomas and lymphomas in contrast to epithelial lesions with presently undefined biological implications.

Fatty acylation of the rat asialoglycoprotein receptor. The three subunits from active receptors contain covalently bound palmitate and stearate.

Rat hepatic asialoglycoprotein receptors (ASGP-Rs) are hetero-oligomers composed of three homologous glycoprotein subunits, designated rat hepatic lectins (RHL) 1, 2, and 3. ASGP-Rs mediate the endocytosis and degradation of circulating glycoconjugates containing terminal N-acetylgalactosamine or galactose, including desialylated plasma glycoproteins. We have shown in permeable rat hepatocytes that the ligand binding activity of one subpopulation of receptors (designated State 2 ASGP-Rs) can be decreased or increased, respectively, by ATP and palmitoyl-CoA (Weigel, P. H., and Oka, J. A. (1993) J. Biol. Chem. 268, 27186-27190). We proposed that a reversible and cyclic acylation/deacylation process may regulate ASGP-R activity during endocytosis, receptor-ligand dissociation, and receptor recycling. In the accompanying paper (Zeng, F-Y., and Weigel, P. H. (1995) J. Biol. Chem. 270, 21388-21395), we show that the ligand binding activity of affinity-purified State 2 ASGP-Rs is decreased by treatment with hydroxylamine under mild conditions consistent with these ASGP-Rs being fatty acylated in vivo. In this study, we used a chemical method to determine the presence of covalently-bound fatty acids in individual ASGP-R subunits. The affinity-purified ASGP-R preparations were separated by SDS-polyacrylamide gel electrophoresis under nonreducing conditions, and the gel slices containing individual RHL subunits were treated with alkali to release covalently bound fatty acids, which were subsequently analyzed by gas chromatography and confirmed by gas chromatography-mass spectrometry. Both stearic and palmitic acids were detected in all three receptor subunits. Pretreatment of ASGP-Rs with hydroxylamine before SDS-polyacrylamide gel electrophoresis reduced the content of both fatty acids by 66-80%, indicating that most of these fatty acids are attached to cysteine residues via thioester linkages. Furthermore, when freshly isolated hepatocytes were cultured in the presence of [3H]palmitate, all three RHL subunits in affinity-purified ASGP-Rs were metabolically labeled. We conclude that RHL1, RHL2, and RHL3 are modified by fatty acylation in intact cells.

Differential response of the epidermal growth factor receptor tyrosine kinase activity to several plant and mammalian lectins.

Biosignalling via lectins may involve modulation of protein kinase activities. This aspect of the biological action of mammalian and plant lectins has been investigated for their effect on the activity of the isolated epidermal growth factor receptor (EGFR). The constitutive tyrosine kinase activity of the epidermal growth factor receptor from rat liver, isolated by calmodulin-affinity chromatography, was activated by concanvalin A (ConA), and wheat germ agglutinin (WGA) to a similar extent as the measured enhancement induced by EGF. In contrast, two mannose-specific lectins, the mannan-binding protein (MBP) and serum amyloid P component (SAP), isolated from human serum, have inhibitory effects, both in the absence and presence of EGF. The differential effects of these lectins were tested using as phosphorylatable substrates a co-polymer of glutamic acid-tyrosine, as well as calmodulin. However, two galactoside-specific lectins, the laminin-binding beta-galactoside-binding 14 kDa lectin, isolated from bovine heart (14K-BHL), and the alpha/beta-galactoside-binding lectin, isolated from mistletoe (Viscum album L.) leaves (VAA), do not inhibit the EGFR tyrosine kinase activity. The sugar dependence of the lectin-mediated action was studied by inhibition assays. Mannose and a mannose-containing neoglycoprotein prevent the activating effect of ConA, and N-acetyl-D-glucosamine partially prevents the activation produced by WGA. However, mannose and mannose-containing neoglycoprotein were ineffective to reduce the inhibitory effect of MBP or SAP.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the macrophage migration inhibitory factor-binding site of sarcolectin and its relationship to human serum albumin.

The sialic acid-binding protein sarcolectin from human placenta specifically interacts with the lymphokine macrophage migration inhibitory factor, enabling its convenient purification and histochemical localization. After cyanogen bromide-mediated cleavage of sarcolectin one polypeptide with an apparent molecular weight of approximately 15,000 exhibited binding capacity to the labelled lymphokine, as revealed by ligand blotting. The N-terminal sequence stretch of this peptide is identical to the respective sequence of human serum albumin, following the internal methionine residue in position 298. Cleavage at a methionine moiety in position 446 can explain the size of the 15 KDa product of chemical degradation. Close similarity of circular dichroism of sarcolectin and human serum albumin added further evidence to their structural similarity, calling for further studies to rigorously define their relationship.

Correlation of expression of binding sites for synthetic blood group A-, B- and H-trisaccharides and for sarcolectin with survival of patients with bronchial carcinoma.

Carrier-immobilised carbohydrates were used to monitor the presence of specific carbohydrate-binding sites in tissue sections. Sarcolectin, an interferon-alpha/beta antagonist and growth regulator, had been shown to bind the lymphokine macrophage migration inhibitory factor (MIF). The lectin is thus a MIF-specific probe. Biotinylated sarcolectin, neoglycoproteins with lactose or N-acetylglucosamine residues, and polyacrylamide-attached trisaccharides, that represent the ABH histo-blood group antigens, were applied to sections of 187 primary lung carcinomas. The panel of cases consisted of 57 epidermoid carcinomas, 55 adenocarcinomas, 43 large cell anaplastic carcinomas and 32 small cell anaplastic carcinomas. 47 cases with intrapulmonary metastatic tumours were also included. Expression of binding sites of both sarcolectin and trisaccharides of histo-blood group antigens A and H correlated with patient survival in lung cancer. In view of the widely performed analysis of the presence of histo-blood group antigens, concomitant profiling of binding sites for these sugar components is suggested to be of potential benefit.

Detection of the lymphokine migration inhibitory factor in normal and disease-affected lung by antibody and by its major binding protein, the interferon antagonist sarcolectin.

Human migration inhibitory factor (MIF) is suggested to play a notable role in regulation of macrophage functions in host defense. A major binding component for the lymphokine in human tissue is the interferon antagonist sarcolectin. This high-affinity interaction gives access to MIF by affinity chromatography on immobilized sarcolectin and may be of significance for in situ activity of MIF. Localization of MIF is one step towards answering this question. Labelled sarcolectin and MIF-specific antibodies can be employed to analyze the expression of the factor. Surgical specimens of 74 patients, who underwent lobe/lung resection or diagnostic biopsy, were fixed with buffered formalin and embedded in paraffin. The material consisted of 36 cases of morphologically normal lung parenchyma of patients, suffering from bronchial carcinoma, of 16 cases with sarcoidosis, of 15 cases with tuberculosis and of 7 cases with idiopathic interstitial pneumonitis. The two types of probe to visualize presence of MIF invariably showed the same level of reactivity, underscoring the potential physiological significance of sarcolectin-MIF interaction. In detail, all cases with pneumonitis, most tuberculosis-affected as well as normal cases and 44% of the cases with sarcoidosis were positive. All positive cases with sarcoidosis and some cases from the other groups revealed accessible binding sites for biotinylated MIF.

Alteration of human lung parenchyma associated with primary biliary cirrhosis.

The clinical history, radiological and histomorphological alterations of the lung parenchyma in a patient suffering from primary biliary cirrhosis are described. The 70-year-old woman had developed a primary biliary cirrhosis, verified by serological abnormalities (AMA positivity, elevated IgM levels) and by liver biopsy. The lung parenchyma displayed immature epithelioid granulomas and characteristics of a chronic organizing pneumonia. Lung function revealed moderate restrictive changes; chest radiographs revealed bilateral, diffuse, patchy infiltrates in the basal lobes. Application of immunohistology detected antigens in liver cells reactive with anti-IgD and anti-IgG, in pneumocytes those reactive with anti-IgD. Presence of macrophage migration inhibitory factor (MIF) by application of its antibody and of the ligand sarcolectin as well as expression of binding capacities to MIF could not be demonstrated in the liver and lung parenchyma. Neoglycoproteins exposing fucose, N-acetyl-D-glucosamine, lactose and mannose residues did not bind to both the liver and lung tissue. The data indicate that at least some patients with primary biliary cirrhosis may develop or suffer from immunological abnormalities, affecting the lung.

Carbohydrate-binding specificity of calcyclin and its expression in human tissues and leukemic cells.

Binding of biotinylated fetuin in a solid-phase assay served as activity assay for purification of calcyclin, the product of a cell growth-related cDNA with homologies to Ca(2+)-binding proteins. Asialofetuin failed to bind to calcyclin, emphasizing the importance of sialic acids. Binding of fetuin was most effectively reduced by N-glycolylneuraminic acid within a panel of mostly negatively charged sugars. Bovine submaxillary mucin and the ganglioside GM1, but not asialo-GM1, proved more effective than neoglycoproteins, carrying negatively charged carbohydrate moieties. Extension of N-acetyl-neuraminic acid to its lactosyl derivative increased its inhibitory potency. Among charge-free carbohydrate residues, only N-acetylglucosamine, lactose, and mannose, but not fucose, melibiose, or N-acetylgalactosamine affected fetuin binding, substantiating the inherent selectivity. Chemical modification with group-specific reagents revealed that lysine and arginine residues appear to be involved in ligand binding that is optimal in the presence of Ca2+, but not Zn2+ and stable up to 1 m NaCl. Biotinylation of calcyclin by modification of carboxyl groups facilitated performance of solid-phase assays with calcyclin in solution, yielding similar results with (neo)glycoproteins in relation to assays with immobilized calcyclin, thereby excluding an impact of binding to nitrocellulose on calcyclin's specificity. Subcellular fractionation disclosed the presence of fetuin-binding activity in all fractions, the specific activity decreasing from the nuclear to the particulate cytoplasmic fraction and the cytoplasmic supernatant. Affinity-purified antibodies were employed to detect high levels of calcyclin expression in acute lymphoblastic, myelogenous, and monocytic leukemia cell lines, but not in myeloma or lymphoblastoid cells. In comparison, most cells were nearly devoid of an O-acetylsialic acid-specific protein that is more abundant in various tissue types than calcyclin.