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T Stigbrand

Publications and source records attributed to T Stigbrand.

At least 73 records · Page 4Linked to original sources
PubMed1994

Use of hydrophobic affinity partitioning as a method for studying various conformational states of the human alpha-macroglobulins.

The serum proteins alpha 2-macroglobulin and pregnancy zone protein undergo major conformational changes when complexed with proteinases. It is shown that the changes in delta log Kmax determined by hydrophobic affinity partitioning is a measure of the extent of changes in the conformation of these alpha-macroglobulins. We introduce a new term for the changes of surface hydrophobicity in a protein as delta log Kacc. This defines the difference of delta log Kmax between a modified and an unmodified conformational state of a specific protein and can be useful as a parameter to describe the apparent conformational changes in the protein.

Centrifugation↗
PubMed1994

Binding of transforming growth factor-beta (TGF-beta) to pregnancy zone protein (PZP). Comparison to the TGF-beta-alpha 2-macroglobulin interaction.

Pregnancy zone protein (PZP) is quantitatively the most important pregnancy-associated plasma protein and it has strong similarity to alpha 2-macroglobulin. Since alpha 2-macroglobulin is a binding protein for transforming growth factors-beta (TGF-beta), it was of interest to test whether the related protein, PZP, also binds to these growth-regulatory proteins. Using affinity-labelling methods, we demonstrate that PZP binds both TGF-beta 1 and TGF-beta 2 and that the binding characteristics are similar to those of the TGF-beta-alpha 2-macroglobulin interaction. TGF-beta 2 and TGF-beta 1 bind to PZP in a predominantly noncovalent manner in vitro. TGF-beta 1 and TGF-beta 2 bind to both the dimeric and tetrameric forms of PZP. Our studies also indicate that PZP binds TGF-beta 2 with higher affinity than TGF-beta 1. Finally, we demonstrate that PZP inhibits the binding of TGF-beta 1 and TGF-beta 2 to their cell surface receptors. The increased level of PZP during pregnancy may affect the action of TGF-beta by regulating the distribution, clearance and/or general availability of TGF-beta. The preferential binding of TGF-beta 2 over TGF-beta 1 by PZP implies that PZP may differentially regulate the action of TGF-beta 1 and TGF-beta 2.

Autoradiography↗
PubMed1994

Correlation of beta-camera imaging and immunohistochemistry in radioimmunotherapy using 90Y-labeled monoclonal antibodies in ovarian cancer animal models.

Tumor stroma contains much fibrin and monoclonal antifibrin antibody targeting is possible in tumors. In this study, nude mouse human ovarian carcinoma xenograft specimens were investigated after treatment with 90Y-labeled monoclonal antifibrin antibody Fab fragment or with 90Y-labeled OC125-monoclonal antibody F(ab')2 fragments. The mice received the radioimmunotherapy activity either intratumorally, intraperitoneally, or intravenously. Beta-camera imaging (BCI) is a novel device for studying activity distribution in tissue specimens and, together with immunohistochemistry (IHC) with OC125, antifibrin, anticarcinoembryonic antigen, anti-cytokeratin, and anti-placental alkaline phosphatase antibodies, was used for correlation of activity distribution of tissue specimens. These results were in concordance: Antigen distribution measured with IHC and radioactivity distribution were similar with the same antibodies, antifibrin, and OC125: However, these antigens demonstrated rather different distribution. Tissue studies revealed that activity was concentrated also in the necrotic tumor tissue, indicating that cell death was also caused by radiation. Differences in the tumor cell morphology were observed using different routes of administration. With BCI, it is possible to quantitate activities in frozen sections (microdosimetry), and these results were in concordance with absolute activities as measured by tissue sampling and well-counting. Three-dimensional reconstruction of tissue slices combined with radioactivity distribution measured with BCI allows estimation of total absorbed radiation dose in tumor after an appropriate dose planning.

Animals↗
PubMed1994

Immunolocalization of stratum corneum chymotryptic enzyme in human skin and oral epithelium with monoclonal antibodies: evidence of a proteinase specifically expressed in keratinizing squamous epithelia.

Stratum corneum chymotryptic enzyme (SCCE) is a recently discovered serine proteinase, which has been purified from human plantar stratum corneum. Evidence has been presented that it may play a role in the terminal stages of epidermal turnover, especially in desquamation. Two mouse monoclonal antibodies (MAb) were raised, TE4b and TE9b, that reacted specifically with SCCE in immunoprecipitation, immunoblotting, and gel-exclusion chromatography. When used in immunohistochemical experiments with the peroxidase-anti-peroxidase method, both MAb detected an antigen located in high suprabasal keratinocytes of the epidermis in normal human skin and at the vermilion border of the lip, with maximal staining of the stratum granulosum. In the hair follicles the MAb reacted with the inner root sheet only. In human oral mucosa the MAb stained the high suprabasal epithelial cells of the hard palate. This is a site where the epithelium forms an orthokeratotic stratum corneum. There was no specific staining of the epithelium of the lip mucosa or the buccal mucosa, where the epithelium does not form a stratum corneum under non-pathological conditions. A correlation therefore seems to exist between the presence of SCCE in high suprabasal cells and the ability of the epithelium to form an orthokeratotic cornified layer. We suggest that SCCE is specifically expressed in keratinizing squamous epithelia and that its expression may be part of the terminal differentiation program of this type of epithelium. These results also give further support to the idea that SCCE may play a role in the turnover and/or formation of the stratum corneum.

Cell Differentiation↗
PubMed1993

Probing different conformational states of pregnancy-zone protein. Fluorescence studies utilizing the binding of 4,4'-bis(8-anilino-1-naphthalenesulphonate).

The binding of the fluorescence probe 4,4'-bis(8-anilino-1-naphthalenesulphonate) (bis-ANS) to the human proteinase inhibitor pregnancy-zone protein (PZP) and its complexes with methylamine and chymotrypsin were investigated. The existence of dimeric PZP-chymotrypsin complex was demonstrated and both the dimeric and the tetrameric PZP-chymotrypsin complexes could be studied separately. The fluorescence data indicate that bis-ANS binds to two different sites on PZP and its complexes. The values of the dissociation constant, Kd1, for the binding to the high-affinity site were determined to be 231 +/- 14, 220 +/- 28, 114 +/- 15 and 49 +/- 1 nM, for the binding to native PZP, PZP-methylamine and dimeric and tetrameric PZP-chymotrypsin, respectively. An 11-30-fold decrease was observed in the affinity for the second site, the corresponding values of the dissociation constant, Kd2, being 1.5-2.8 +/- 1.0 microM, which are not significantly different for PZP and its derivatives. The results suggest that the probe bis-ANS discriminates between the different conformational states of PZP and that while the conformation of the complex with methylamine does not differ much from that of the native protein, there is a significant change in conformation when chymotrypsin cleaves the bait region. This is substantiated by a 30%-45% decrease in the maximum enhancement of fluorescence intensity when PZP is treated with chymotrypsin. Although the dimeric and tetrameric forms of PZP-chymotrypsin complexes differ in Kd1 values, the difference in the maximum enhancement of the fluorescence of bis-ANS by the two forms is not significant. This indicates that dimer-dimer interaction in the tetrameric form does not involve hydrophobic sites. The necessity of bait-region cleavage for extensive conformational changes in PZP distinguishes it from alpha 2-macroglobulin, the other alpha-macroglobulin in human plasma.

Anilino Naphthalenesulfonates↗
PubMed1993

Comparison of conformational changes of pregnancy zone protein and human alpha 2-macroglobulin, a study using hydrophobic affinity partitioning.

Conformational changes of human alpha 2-macroglobulin (alpha 2M) and pregnancy zone protein (PZP), reflected in changes in surface hydrophobicity, have been studied. The results show that the conformation of alpha 2M is governed by the degree of 'trapping'. Thus, cleavage in the bait region and of the thiol ester by proteinase treatment causes a two-fold increase in surface hydrophobicity of alpha 2M. However, the increase is still higher (three-fold) when the thiol esters in alpha 2M alone are cleaved by methylamine. Cyanylation of the thiol groups exposed upon methylamine treatment yields a derivative with the same hydrophobicity as native alpha 2M. Treatment of this derivative with chymotrypsin restores the hydrophobicity to that of methylamine-treated alpha 2M. Since the C-terminal 18 kDa fragment of alpha 2M exhibits no hydrophobicity, the change in hydrophobicity seems not to reside in the receptor binding site. In contrast to alpha 2M, modification of both native and methylamine-treated PZP with chymotrypsin gives a reduction (about 40%) in hydrophobicity. The change in hydrophobicity is insignificant on treatment with methylamine alone. Furthermore, hydrophobic interactions appear not to contribute to tetramerization of PZP. The present study indicates major differences in the conformational states of alpha 2M and PZP as reflected in the hydrophobic surfaces exhibited.

Chymotrypsin↗
PubMed1993

Production of conformation-specific monoclonal antibodies against alpha 2 macroglobulin and their use for quantitation of total and transformed alpha 2 macroglobulin in human blood.

Monoclonal antibodies against the human proteinase inhibitor, alpha 2 macroglobulin, have been produced by immunizing BALB/c mice with alpha 2 macroglobulin reacted with methylamine. Two antibodies have been characterized in detail with respect to their binding to native alpha 2 macroglobulin and to different derivatives of the inhibitor. The antibody alpha-1 was found to recognize only those forms of the inhibitor which were transformed by reaction with different proteinases or with methylamine. Binding of alpha-1 was mapped to a specific epitope localized within a distance of 138 amino acid residues from the C terminal end of alpha 2 macroglobulin. The C terminal end is assumed to be exposed during the transformation of the inhibitor and harbours the receptor recognition site. The monoclonal antibody alpha-11 was found to bind to all forms of the inhibitor indicating that its epitope is located in a region not involved in major conformational changes of the inhibitor. On the basis of the different reactivity patterns of alpha-1 and alpha-11 two enzyme-linked immunosorption assays were established for quantitation of total and transformed alpha 2 macroglobulin in human blood. The concentration of the two forms have been determined in a population of 114 healthy individuals giving values of 254 +/- 6.6 mg/dl (mean +/- SEM) of total alpha 2 macroglobulin and 1.07 +/- 0.05 mg/dl (mean +/- SEM) of the transformed inhibitor.

Animals↗
PubMed1993

Human intestinal alkaline phosphatase--release to the blood is linked to lipid absorption, but removal from the blood is not linked to lipoprotein clearance.

To evaluate a possible quantitative relationship between the rise in intestinal alkaline phosphatase (IAP) activity and triglyceride-rich lipoproteins in the blood after an oral fat intake, a specific and sensitive immunocatalytic assay was used. First, day to day variation of the basal IAP activity in the blood of eight volunteers was evaluated. One group of subjects with high basal IAP activity and great variations from one day to the other was distinguished from a group with low basal IAP activity and small day to day variations. The subjects with high basal IAP activities were all secretors of red blood cell antigens, while those with low basal IAP activities were non-secretors. The IAP activity in the blood rose after fat rich meals but not after fat free meals. To further investigate this, IAP activities were measured in the blood of 28 men before 3, 6, 9 and 12 h after a standardized oral fat load of a mixed meal type. The activity rose in all individuals and showed a similar time course as the rise in plasma TG concentration. The elevations of the IAP activity were, however, not quantitatively correlated with the elevations in TG concentration, nor were there any relations in time to peak levels. Subjects with high basal IAP increased their IAP activity more than those with low basal IAP, but within neither group there was any correlation between the basal IAP activity and the rise in IAP activity. IAP did not float with lipoproteins on ultracentrifugation of plasma, nor did IAP bind to lipid droplets from a fat emulsion added to plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Protein patterns in human vestibular ganglion cells and hair cells, with functional interpretations.

Cytoskeletal organization was analysed in the vestibular ganglion cells and in the sensory epithelia of all five vestibular organs of the adult human temporal bone. A pancellular rigidity of the upper surfaces of the vestibular organs, evidenced by large quantities of intermediate filaments and actin, seems of importance for the mechanoelectrical transduction by opposing the motion of the cupulae and statoconial layers. Immunostaining for the calcium-binding protein synaptophysin in the sensory hair bundles and in the area close to the cuticular plates--the first of its kind to be demonstrated in human vestibular organs--indicates the presence of calcium-dependent ionic channels. The hypothesis is presented that the calyx might be involved in a short-loop feed back control of type I hair cells, i.e. of the mechanoelectrical transduction itself. Subpopulations of vestibular ganglion cells were identified by the staining pattern of cytoskeletal proteins, but not by ordinary ultrastructural analysis.

Actins↗
PubMed1993

Radioimmunodetection of ovarian cancer.

Radioimmunoscintigraphy (RIS) is a potentially valuable method for the detection of primary, secondary and recurrent malignant tumours. Antigens that have been used for monitoring as well as for RIS of ovarian carcinomas include CA 125, PLAP, HMFG, and CA 19-9. Between 70 and 100% of the tumours have been detected at RIS when these antigens have been used. Conventional methods, e.g., computerized tomography (CT) and ultrasonography (US), demonstrate similar or lower detection rate than RIS for tumour diagnosis. RIS gives additional information to conventional radiological methods (CT and US) for the detection of occult ovarian carcinomas. A review of earlier investigations is given and our own recent results using PLAP as a target antigen are presented. The future potential of the technology is discussed.

Adult↗
PubMed1993

Alternative technologies to generate monoclonal antibodies.

Several new technologies to generate and modify established hybridomas that produce monoclonal antibodies have recently been presented and further development should make them more suitable for diagnostic and therapeutic techniques. Different proteolytic procedures have been used for the fragmentation of intact antibodies to Fab2' and Fab fragments and recombinant DNA techniques have made it possible to obtain chimeric, humanized, Fv fragments and single-chain Fvs. A review of the new approaches is presented and the future implications are discussed.

Animals↗
PubMed1992

Differences in the proteinase inhibition mechanism of human alpha 2-macroglobulin and pregnancy zone protein.

Different conformational states of human alpha 2-macroglobulin (alpha 2M) and pregnancy zone protein (PZP) were investigated following modifications of the functional sites, i.e. the 'bait' regions and the thiol esters, by use of chymotrypsin, methylamine and dinitrophenylthiocyanate. Gel electrophoresis, mAb (7H11D6 and alpha 1:1) and in vivo plasma clearance were used to describe different molecular states in the proteinase inhibitors. In alpha 2M, in which the thiol ester is broken by binding of methylamine and the 'trap' is closed, cyanylation of the liberated thiol group from the thiol ester modulates reopening of the 'trap' and the 'bait' regions become available for cleavage again. The trapping of proteinases in the cyanylated derivative indicates that the trap functions as in native alpha 2M. In contrast, cyanylation has no effect on proteinase-treated alpha 2M. As demonstrated by binding to mAb, the methylamine and dinitrophenylthiocyanate-treated alpha 2M exposes the receptor-recognition site, but the derivative is not cleared from the circulation in mice. The trap is not functional in PZP. In native PZP and PZP treated with methylamine, the conformational states seem similar. The receptor-recognition sites are not exposed and removal from the circulation in vivo is not seen for these as for the PZP-chymotrypsin complex. Tetramers are only formed when proteinases can be covalently bound to the PZP. Conformational changes are not detected in PZP derivatives in which the thiol ester is treated with methylamine and dinitrophenylthiocyanate. The results suggest that the conformational changes in alpha 2M are generated by mechanisms different to these in PZP. The key structure gearing the conformational changes in alpha 2M is the thiol ester, by which the events 'trapping' and exposure of the receptor-recognition site can be separated. In PZP, the crucial step for the conformational changes is the cleavage of the 'bait' region, since cleavage of the thiol ester does not lead to any detectable conformational changes by the methods used.

Animals↗
PubMed1992

Placental alkaline phosphatase has a binding site for the human immunoglobulin-G Fc portion.

Affinity chromatography of human plasma on placental-alkaline-phosphatase-Sepharose columns (placental alkaline phosphatase, PLAP) yielded consistently a pure protein which was identified as IgG on the basis of electrophoretical and immunological comparisons with authentic human IgG. SDS/PAGE of the protein revealed, under reducing conditions, two polypeptides of 55 kDa and 25 kDa. The N-terminal amino acid sequence (12 residues) of the 55-kDa subunit presented high similarity (83-100%) with known sequences of immunoglobulin gamma chains. The IgG binds by its Fc portion to a fully exposed domain in the plasma-membrane-anchored PLAP. Scatchard analysis of the interaction gave a dissociation constant of 3.68 microM, a value close to those found for haematopoietic cells and syncytiotrophoblast Fc receptors. The latter was affinity purified from human placenta as the major IgG-binding component and presented cross-immunoreactivity with anti-PLAP antibodies, indicating that PLAP and the putative placental Fc receptor could be identical molecules.

Alkaline Phosphatase↗
PubMed1992

Placental alkaline phosphatase, a GPI-anchored protein, is clustered in clathrin-coated vesicles.

Pure clathrin-coated vesicles were prepared from a fresh human placenta. The analysis of their content revealed the presence of transferrin, low density lipoproteins, IgG and placental alkaline phosphatase. Since the latter is a membrane protein anchored by a glycan-phosphatidyl inositol (GPI) moiety, its presence in coated vesicles was unexpected. Placental alkaline phosphatase is neither adsorbed to the surface of the vesicles, nor appearing due to plasma membrane contaminants, but is located in the lumen of the vesicles. The presence of alkaline phosphatase in coated vesicles strengthens its postulated physiological role in the transcytosis of IgG molecules in placenta.

Alkaline Phosphatase↗
PubMed1992

Interactions of human alkaline phosphatase isoenzymes with triazine dyes using affinity partitioning, affinity chromatography and difference spectroscopy.

Aqueous two-phase systems consisting of dextran, polyethylene glycol and dye-liganded polyethylene glycol were employed to investigate the affinity partitioning behaviour of isoenzymes of human alkaline phosphatase. Whereas in the system without a dye ligand the partition coefficients of the isoenzymes from human intestine and placenta were identical, the isoenzyme from human liver showed a significantly lower partition coefficient under the same conditions. After addition of dye-liganded polyethylene glycol two groups of dyes possessing substantial affinities to the isoenzymes were found. One, represented by Procion Yellow HE-3G, interacts specifically with the active centre of the isoenzymes. Differences in the affinity of the isoenzymes towards the individual dye ligands are caused only by the carbohydrate content, especially by the terminal sialic acid residues. The other group of dye ligands, represented by Procion Navy MX-RB, binds obviously in a more complex fashion involving other binding sites, which are only present in alkaline phosphatase of human liver. Procion Navy MX-RB was found to function as a suitable affinity ligand for the separation of human liver alkaline phosphatase from the other isoenzymes. Differences in the primary structure of two allelic forms of human placental alkaline phosphatase [(SS) and (F)] are not recognized in aqueous two-phase systems with or without dye-liganded polyethylene glycol.

Alkaline Phosphatase↗
PubMed1992

Placental alkaline phosphatase is related to human IgG internalization in HEp2 cells.

The biological function(s) of placental alkaline phosphatase has not yet been unraveled. The low catalytic activity of the enzyme at physiological pH, and the lack of "natural substrates", bring about the necessity of a more structure-related conception of its role. We have observed an interaction between placental alkaline phosphatase and human IgG. In this report we show that this isozyme is the major membrane protein able to bind IgG in a IgG-internalizing cell line (HEp2). Pretreatment of these cells with Fab fragments of anti placental alkaline phosphatase antibodies blocks the internalization of IgG without perturbing the endocytosis of other ligands. Our results indicate that placental alkaline phosphatase has the ability not only to bind human IgG, but also to promote its internalization in HEp2 cells.

Alkaline Phosphatase↗