Biomedical subjects

T Stigbrand

Publications and source records attributed to T Stigbrand.

At least 37 records · Page 2Linked to original sources

PubMed1999

Stability and immunoreactivity of the monoclonal anticytokeratin antibody TS1 after different degrees of iodination.

The immunoreactivity, stability and in vivo kinetics of an anticytokeratin 8 monoclonal antibody, TS1, were investigated following different degrees of labeling with 125I (0.2, 1 and 2-3 125I/TS1 MAb). By testing with ELISA, it was demonstrated that a high degree of iodination, i.e. > 2 125I/TS1, caused a rapid decrease in immunoreactivity to almost zero within 10 days. Furthermore, a complete degradation to low molecular weight fragments and free iodine was seen, as shown by SDS PAGE and autoradiography. The differently labeled radionuclide conjugates were injected into nude mice inoculated with HeLa Hep2 cells and tumor doses (estimated by MIRD formalism), tumor:non-tumor dose ratios, % I.D./gram tissue, Gy/MBq and in vivo kinetics of the differently labeled MAbs were determined. Despite the in vitro instability of the highest iodinated radionuclide conjugate, it was possible to deliver high doses to the tumors if the conjugate was injected into the animal immediately after completion of the iodination procedure. Increases from 1.4 Gy to 15.2 Gy delivered tumor dose were obtained with a tenfold increase in the specific activity, without alterations in the tumor:non-tumor tissue dose ratios. There is room for significant improvements in efficacy at radioimmunotherapy, which can be gained by optimizing the degree of iodination. For therapeutical applications a high degree of iodination may be an advantage.

Animals↗

PubMed1999

Radioimmunotherapy of DU-145 tumours in nude mice--a pilot study with E4, a novel monoclonal antibody against prostate cancer.

The anti-tumour effect of the 131I-labelled antiprostate monoclonal antibody (MAb) E4 was studied in an experimental model with 41 nude mice, subcutaneously xenografted with a human prostate cancer cell line (DU-145). The mice were divided into four study groups, i.e. one receiving single and another repeated injections of the radiolabelled MAb. A third group was injected with non-labelled MAb, and the fourth served as an untreated control group. The tumour volumes increased similarly in all groups during the 27-day observation period. The tumour tissue was morphologically disintegrated in the group that received repeated radioimmunotherapy (RIT). The tumours from this group contained large fluid-filled cystic parts and demonstrated pronounced cellular and subcellular polymorphism in the remaining viable tumour tissue. The untreated control tumours and single therapy tumours remained solid. The proportion of the total tumour volume that consisted of viable tumour cells, as determined by morphometric techniques, was significantly lower in the 131I-E4-treated groups. The use of 131I-labelled E4 MAb has thus demonstrated a promising therapeutic potential.

Animals↗

PubMed1999

Idiotypic-anti-idiotypic antibody interactions in experimental radioimmunotargeting.

Idiotypic-anti-idiotypic antibody interactions can be used in vivo to regulate the serum levels of specific radiolabeled antibodies. Anti-idiotypic antibodies can also be used as clearing agents for radiolabeled antibodies in radioimmunolocalization and radioimmunotherapy. The present study describes the immunochemical interactions between the monoclonal idiotype (H7) and three generated monoclonal anti-idiotypic antibodies (alphaH7:1, alphaH7:35, and alphaH7:38). An unexpected variability in complex formation could be demonstrated in vitro, revealing three different stable complex patterns, i.e., low molecular weight 1:1 complexes, ladder formation with oligomeric, consecutively added constituents, and large linear polymeric complexes of high molecular weight. Within 24 h, the anti-idiotypes were able to cause a significant decrease in total body radioactivity, and the antibody generating a ladder formation (alphaH7:38) was found to be the most efficient at removing radiolabeled idiotypes from the circulation. It is concluded that monoclonal anti-idiotypic antibodies may be valuable tools in improving radioimmunolocalization and radioimmunotargeting.

Animals↗

PubMed1998

Changes in intestinal alkaline phosphatase isoforms in healthy subjects bearing the blood group secretor and non-secretor.

We found the high molecular mass intestinal alkaline phosphatase (HIAP) and normal molecular mass intestinal alkaline phosphatase (NIAP) in serum at fasting and after fatty meal by use of 6.0% polyacrylamide gel electrophoresis (PAGE) in the presence of 1% Triton X-100. HIAP only appeared in serum of Lewis blood group secretors ¿Le(a-b+)¿, and HIAP levels were dependent on ABO blood groups. Among the secretors, the highest activities of HIAP in fasting serum were observed in subjects with blood groups O and B (8.6+/-1.4 U/1; mean+/-SD) and the lowest activities were associated with blood group A (0.7+/-0.2 U/1; mean+/-SD), and the HIAP activities did not change after fatty meal. In contrast, NIAP was present in the serum of both secretors and non-secretors regardless of ABO blood group. Trace amounts of NIAP remained in fasting serum; however serum NIAP activities rose sharply after fatty meal. The remaining ratios of NIAP activity at fasting and 9 h after fatty meal of secretors were approximately the same as those of non-secretors. The electrophoretic mobility on PAGE or the apparent molecular mass estimated by gel filtration of serum NIAP in secretors was slightly different from that in non-secretors. In addition, HIAP can be normalized to NIAP on PAGE in the absence of Triton X-100, and the electrophoretic mobility of normalized-NIAP was identical to that of original NIAP in secretors. Accordingly, it can be concluded that the structure of serum NIAP in the secretor was different from that in the non-secretor, because HIAP is only formed by serum NIAP in the secretor. These results suggest that differences in serum NIAP in the secretor and the non-secretor may be closely related to the appearance of IAP in the circulation.

ABO Blood-Group System↗

PubMed1998

Epitope specificity of 30 monoclonal antibodies against cytokeratin antigens: the ISOBM TD5-1 Workshop.

The epitope specificities of 30 monoclonal antibodies (MAbs) against the most common human cytokeratins. i.e., Nos. 8, 18, and 19, in epithelial cells were investigated in the ISOBM TD-5 Workshop. Seven research groups from universities or companies participated independently in the evaluation of the antibody specificities. The complex assembly of cytokeratins in vivo, with obligatory heterologous dimeric combinations of different cytokeratins from each of the two major groups, comprising together more than 20 different individual cytokeratins, made analysis of the antibody reactivity patterns with isolated single cytokeratins necessary. The concordance of the evaluations was striking and independent of the technologies used. As antigens purified individual cytokeratins, chemically degraded purified cytokeratins, recombinant intact and truncated cytokeratins, as well as specific synthesized shorter peptides were used. In order to elucidate the epitope specificity, reactivity patterns in ELISA assays and immunoblots with partial enzymatic degradation of the antigens were performed. Competitive cross-inhibition experiments between antibodies using antigens and antibodies in all possible combinations were performed with radioimmunometric assays, BIAcore, and ELISA technology. All 30 antibodies could convincingly be classified with regard to target cytokeratin. One MAb (192) had to be deleted due to dual specificities in both isotype and epitope specificity against its target. Six antibodies bound selectively to cytokeratin 8, 14 to cytokeratin 18, and 10 to cytokeratin 19, as demonstrated by using native, recombinant, and synthesized antigens. The immunodominant part of the molecule for all three types of cytokeratins was located in the region of amino acid (aa) 270-400. Out of the six MAbs reactive with cytokeratin 8, four MAbs, i.e., 178, 199, 202, and 206, were reactive with a sequence in the interval aa 340-365, and MAb 191 reacted with a closely related epitope. The remaining antibody, 192, presented dual specificities. At least two closely related major immunogenic epitopes could be identified in cytokeratin 8. In cytokeratin 18 four distinct epitopes could be documented, again with the dominating sequence region 270-429 as target for 10 (181, 184, 186, 188, 189, 190, 193, 196, 198, and 200) out of 14 antibodies. Since MAb 193 is known to react with the M3 epitope, aa 322-342 in cytokeratin 18, this entire group is reactive in the region close to the charge shift, in the middle of the rod 2B region, as shown by competitive binding. The remaining four anticytokeratin 18 antibodies (180, 185, 203, and 205) displayed unique, noncompetitive binding to this filament. Cytokeratin 19, reactive with altogether ten antibodies, displayed two major epitopes, all of them also within the large immunodominant region. MAbs 179, 195, 197, and 204 were reactive with the peptides aa 311-335 also known as the KS 19.1 epitope, and MAbs 182, 183, 187, 194, and 201 bound to peptide aa 346-367, known as the BM 19.21 epitope. One antibody, 231, was selectively reactive with aa 356-370 in cytokeratin 19. A complex pattern of binding specificities comprising at least ten different, noncompetitive epitopes, mainly situated in the rod portion, 2A and 2B, situated close to the charge shift in the rod of all three cytokeratins was documented. Out of the 29 classifiable antibodies, altogether 22 were reactive in this very short region, i.e., from aa 311 to 370 in all cytokeratin filaments. The remaining seven antibodies displayed unique binding properties. The implications of the findings are of significance both for immunohistochemistry and for assaying circulating heterodimeric, partially degraded complexes in patients' blood for tumor marker evaluation.

Amino Acid Sequence↗

PubMed1998

Properties of high-molecular-mass placental alkaline phosphatases in normal pregnancy sera.

We examined the appearance of high-molecular-mass placental alkaline phosphatases (HPLAPs) in the serum of normal pregnant women by means of polyacrylamide gel electrophoresis (PAGE) in the presence of Triton X-100. The HPLAPs were undetectable or only slightly detectable by PAGE in the absence of Triton X-100. The HPLAPs were detected in all sera sampled during the last trimester of pregnancy. The catalytic activities of total placental alkaline phosphatase (TPLAP) and HPLAPs were correlated (r = 0.96) and the ratio of HPLAPs/TPLAP catalytic activity was 0.20 (0.06) (mean and SD) in 40 serum samples from pregnant women. The HPLAPs appear to be formed from a common dimeric placental alkaline phosphatase (PLAP) (common-PLAP), as judged by the fact that they were formed again after removal of HPLAPs from serum by gel filtration. The formation of HPLAPs was more prominently observed with the faster fractions of gel filtration. The apparent molecular mass of the HPLAPs in pregnancy serum was 720 KDa by gel filtration. HPLAPs were not converted to common-PLAP by phosphatidylinositol-specific phospholipase (PIPL) C and PIPL-D treatments. The HPLAPs were selectively incorporated into liposomes consisting of phosphatidylcholine/cholesterol, and most of the PIPL-D-treated PLAP could from HPLAPs, while a small amount of PLAP could not form HPLAPs. On the other hand, HPLAPs in pregnant women's sera and HPLAPs prepared from partially purified PLAP in vitro could be converted to common-PLAP by brief treatment with subtilisin. However, the highly purified PLAP could not form HPLAPs in the presence of Triton X-100. These results suggest that PIPL-D-resistant and PLAP-associated serum protein may regulate the conversion of PLAP to HPLAP in the presence of Triton X-100.

Alkaline Phosphatase↗

PubMed1997

Radioimmunoscintigraphy with a novel monoclonal antiprostate antibody (E4): an experimental study in nude mice.

BACKGROUND: Prostate cancer is one of the leading causes of death among men, despite achievements in diagnosis and therapy. Radioimmunolocalization and radioimmunotherapy of malignant tumors have demonstrated increasing potential and may become useful tools in the management of prostate cancer. METHODS: Nude mice were inoculated subcutaneously with cells from the poorly differentiated human prostate cancer cell line DU-145. The intact monoclonal antibody (MoAb) E4 and an intact anticytokeratin-8 MoAb, TS1, used for comparison were labeled with 125I and injected intraperitoneally (i.p.) in the mice. Repetitive quantitative scintigraphic recordings were performed during 1 month. The mice were killed at Day 29 after injection of the radiolabeled MoAb. The tumors and the organs were dissected and weighed. The remaining activity was measured in a gamma well counter. One part of the tumor was immediately fixed in Bouin's solution for autoradiography and the other in formaldehyde for microscopy. RESULTS: The study demonstrated significant radioimmunolocalization of the MoAb E4 into the DU-145 prostate tumor tissue in the animal model, with an average radiation dose of 0.08 Gy/MBq in the tumor. TS1 localized preferentially in necrotic parts of the tumor, yielding a tumor dose of 0.02 Gy/MBq. CONCLUSIONS: The MoAb E4 is a promising radiotracer for prostate cancer and may be used in radioimmunotherapy. As in earlier studies, TS1 shows significant radioimmunolocalization into necrotic tumor tissue, which also exists in prostate cancer.

Animals↗

PubMed1997

Endogenous anti-tumor antibody responses in nude mice.

BACKGROUND: Nude mice with xenografted human tumors is the most exploited animal model used to elucidate the efficacy of experimental radioimmunolocalization and radioimmunotherapy. These animals accept transplants and are generally considered immunologically inert with regard to cell-mediated and humoral immune responses against the tumors. METHODS: Nude control mice and mice carrying human HeLa Hep-2 tumor xenografts were studied for appearance of endogenous antibodies following inoculation with tumor cells. The titers of these antibodies were investigated by isotype-specific enzyme-linked immunosorbent assay (ELISA) technologies, fluorescence-activated cell sorter analysis (FACS), BIAcore (Pharmacia Biosensor AB, Uppsala, Sweden) technology, and immunofluorescence. RESULTS: The HeLa Hep-2 cell line was found to be immunogenic in all investigated animals by means of ELISA, FACS, and BIAcore evaluations as well as by immunofluorescence against both tested antigens, placental alkaline phosphatase and cytokeratin 8. Predominantly immunoglobulin M antibodies were induced, but immunoglobulin G isotypes could also be identified. Sera from these tumor-bearing mice were used for immunohistochemistry of the tumor cells. The antibodies seemed to be of low affinity and may be displaced by high-affinity monoclonal antibodies used in radioimmunotargeting. CONCLUSIONS: Nude mice bearing tumor xenografts produce significant amounts of antibodies against these two human tumor-derived antigens. These endogenous antibodies may influence targeting of radiolabeled antibodies. They also have the potential to interfere with the pharmacokinetics of labeled or nonlabeled idiotypic antibodies during experimental immunolocalization.

Alkaline Phosphatase↗

PubMed1997

Dosimetry of fractionated administration of 125I-labeled antibody at experimental radioimmunotargeting.

BACKGROUND: Radiotherapy of solid tumors is preferably performed in fractionated doses. Conversely, radioimmunotherapy with nuclide-carrying antibodies delivers a continuously decreasing low dose rate during a longer time period after a single injection. In the current study, the same total amount of 125I-labeled anticytokeratin monoclonal antibody (MoAb) was administrated in one, three, or ten injections and the dosimetry was evaluated. METHODS: Three groups of nude mice (10 mice each) with HeLa Hep 2 xenografts were injected with 1 x 100 microg/22.2 megabecquerel (MBq), 3 x 33 microg/7.4 MBq, and 10 x 10 microg/2.22 MBq 125I-labeled TS1 MoAb, respectively. The mice were examined scintigraphically over a 54-day period (total number of radio immunoscintigraphies (RISs) = approximately 700) and doses to tumor and normal tissues were estimated according to the medical internal radiation dose formalism. RESULTS: A single bolus injection caused higher tumor uptake, tumor dose, and tumor to nontumor dose ratio than administration of the same total dose of antibody and radioactivity in three or ten separate injections. The single bolus injection caused a tenfold higher tumor uptake (% injected dose, or ID) compared with the group receiving ten injections. This caused a tumor dose of 17 gray to the group receiving a single bolus injection. CONCLUSIONS: In this antigen target system, a single injection of a large amount of antibody was found to be more efficient than the same antibody dose subdivided into three or ten fractions. It was concluded that not only the radioactivity but also the amount of antibody per fraction should be considered when determining optimal fractionated radioimmunotherapy.

Animals↗

PubMed1997

Dosimetry of fractionated experimental radioimmunotargeting with idiotypic and anti-idiotypic anticytokeratin antibodies.

BACKGROUND: Repeated injections of iodine-125 (125I)-labeled tumor targeting anticytokeratin monoclonal antibody (TS1) and a nonlabeled antiidiotypic monoclonal antibody against TS1 (alphaTS1) were compared with a single injection of the radiolabeled TS1 in experimental radioimmunotargeting. Anti-TS1 was used to remove nontargeting TS1. METHODS: Nude mice were inoculated with HeLa Hep2 cells. The animals in Group A received a single injection of 13 MBq 125I-labeled TS1. The animals in Group B received four injections of 125I-labeled TS1 (8-13 MBq) followed by alphaTS1 24 hours later, at 2-week intervals. The mean absorbed doses were calculated according to the Medical Internal Radiation Dose Committee criteria based on repetitive radioimmunoscintigraphies during an observation period of 59 days. RESULTS: A 11 gray (Gy) mean dose to the tumor and 2 Gy to the whole body was achieved in Group A. Mean peak tumor uptake of 5% of the injected dose (ID), corresponding to 14% ID/g, was observed on Day 17 after a single injection of the labeled monoclonal antibody. A mean peak tumor uptake of the same order of magnitude was seen in Group B. An absolute increase in the tumor uptake was observed in Group B during the entire observation period. The mean absorbed dose to the tumors was 11 Gy at the end of the observation period, whereas the whole body dose was only 2.5 Gy in Group B. Autoradiography of the tumors at the end of the observation period confirmed an intensive heterogeneous accumulation of activity in the entire tumor. CONCLUSIONS: The fractionated strategy can contribute to a significant accumulation of radiolabeled TS1 in the tumors. Furthermore, the use of alphaTS1 makes it possible to increase the tumor-to-nontumor dose ratio and maintain a prolonged high activity accumulation in the tumor.

Animals↗

PubMed1997

Experimental radioimmunotargeting combining nonlabeled, iodine-125-labeled, and anti-idiotypic anticytokeratin monoclonal antibodies: a dosimetric evaluation.

BACKGROUND: Preinjection of a nonlabeled tumor targeting anticytokeratin monoclonal antibody (TS1) and postinjection of an anti-idiotypic anticytokeratin monoclonal antibody (alphaTS1) were evaluated separately and in combination to investigate their effects on the accumulation of iodine-125 (125I)-labeled TS1 in experimental radioimmunotargeting. TS1 targets deposited extracellular cytokeratin 8 from necrotic tumor cells. METHODS: Nude mice were inoculated with HeLa Hep 2 cells. Four different groups were followed with 504 repetitive quantitative radioimmunoscintigraphic recordings during a 78-day observation period. The absorbed doses were calculated according to criteria of the Medical International Radiation Dose Committee. RESULTS: As much as 2% of the injected dose (ID) of 125I-labeled TS1 accumulated in the tumor, and the peak tumor uptake was recorded as late as Day 30 after the injection of 125I-labeled TS1. Anti-TS1 caused a rapid decrease in the whole body activity. The highest tumor-to-nontumor activity ratios were obtained when a pre-injection of nonlabeled TS1 was combined with a postinjection of alphaTS1. The mean absorbed dose in tumor per unit activity administered was 0.44 gray/megabecquerel (Gy/MBq) and in nontumor tissues 0.15 Gy/MBq after a single injection of 125I-TS1. The efficacy was 0.34 Gy/MBq in tumor and 0.1 Gy/MBq in nontumor tissues after a combination of preinjection of nonlabeled TS1 and postinjection of nonlabeled alphaTS1. This indicates a 20% increase in tumor doses compared with a single injection of labeled TS1. CONCLUSIONS: This study confirms an extensive accumulation of TS1 in the tumor, with peak values as late as 30 days after injection of labeled TS1. Furthermore, both preinjection of nonlabeled TS1 and postinjection of alphaTS1 can improve radioimmunotargeting.

Animals↗

PubMed1997

Interaction of matrix metalloproteinases-2 and -9 with pregnancy zone protein and alpha2-macroglobulin.

The binding of matrix metalloproteinases-2 and -9 to pregnancy zone protein and alpha2-macroglobulin was studied. The binding was demonstrated by formation of dimeric as well as tetrameric complexes of pregnancy zone protein and by the formation of alpha2-macroglobulin complexes with fast and intermediate mobility in native gel electrophoresis. The complex formation was confirmed by the use of 125I-labeled matrix metalloproteinase-2. The cleavage sites in the "bait" regions following formation of high-molecular-weight complexes of matrix metalloproteinases with the alpha-macroglobulins were determined by protein sequence analysis. Pregnancy zone protein was cleaved at Thr693-Tyr694 and alpha2-macroglobulin at Gly679-Leu680 and Arg696-Leu697 by matrix metalloproteinase-2. Matrix metalloproteinase-9 cleaved alpha2-macroglobulin at the same site as matrix metalloproteinase-2, but cleavage of pregnancy zone protein was at Leu753-Ser754. The sequences of the bands, visualized in the SDS gel, of approximately 90 and 165 kDa or higher molecular weight complexes were the same. This indicates that the matrix metalloproteinases cleaved the inhibitors with or without binding to them. The present results suggest that matrix metalloproteinases-2 and -9 may interact with pregnancy zone protein and alpha2-macroglobulin in vivo.

Animals↗

PubMed1997

Two-dimensional high-resolution electrophoresis of elastin-derived peptides.

Degradation of human aortic elastin in vivo yields a restricted number of differentially sized and charged peptides. Elastin-derived peptides (EDP) can be analyzed by two-dimensional electrophoresis after their extraction from human abdominal aortic tissue by 0.2 M sodium chloride. The peptides were separated according to charge by acetic acid-urea-PAGE and then according to molecular mass by SDS-PAGE. The identity of these peptides as EDP was continued by immunoprecipitation and Western blots. The two-dimensional electrophoretic system can resolve desmosine-like cross-linked EDP of the similar molecular configuration but differing in the number of positively charged amino acid residues. The new separation technique of EDP has the capacity to identify defects in desmosine-like cross-links and may be useful in characterizing abberations in elastin structures.

Animals↗

PubMed1997

The contact zones in human alpha2-macroglobulin--functional domains important for the regulation of the trapping mechanism.

A functional domain termed the contact zone, which is the region of a subunit interacting with another non-covalently bound subunit, is suggested to play a decisive role in the trapping mechanism of human alpha2-macroglobulin. Tetrameric alpha2-macroglobulin can be dissociated into stable dimers with intact thiol esters by sodium thiocyanate, whereby the contact zones are disrupted. The dissociation leads to significant conformational changes, as studied by ultraviolet-difference spectroscopy, CD, fluorescence and affinity partitioning. The conformation of the dimers is similar to that of MeNH2-treated alpha2-macroglobulin, in which the thiol esters are cleaved, a conformational state with a closed trap occurs, and receptor-recognition sites are exposed. The receptor-binding domain is at least partly exposed in the dimer, as judged by binding of specific mAbs. The bait region in the dimers can be cleaved by proteases, and activation of the thiol esters ensues without binding of the protease. When the dimers were treated with MeNH2, no conformational changes could be detected by ultraviolet-difference spectroscopy or CD. The conformational changes occurring on dissociation into dimers are suggested to be related to trap closure and receptor-recognition-site exposure without cleavage of the thiol esters. The model presented here suggests that two separate conformational changes occur in alpha2-macroglobulin upon activation. The first involves changes at the contact zones as a result of the thiol-ester cleavage, and the second causes exposure of the receptor-recognition sites and closure of the trap.

Antibodies, Monoclonal↗

PubMed1997

Purification of pregnancy zone protein and its receptor binding domain from human plasma.

A new significantly improved method for purification of pregnancy zone protein (PZP), alpha 2-macroglobulin (alpha 2M), and the C-terminal PZP receptor binding domain is presented. Several steps in an earlier procedure have been deleted, and modifications in the gradients in the DEAE step leave most of the contaminants bound to a DEAE-Sephacel gel. This procedure makes possible the rapid, simultaneous purification of both of these closely related unstable proteins in native form from human plasma, with no thiolester cleavage or formation of tetrameric PZP. The final preparations of both alpha 2M and PZP are pure as determined by nonreducing and reducing polyacrylamide gel electrophoresis following silver staining and no cross-contamination can be observed. The yield has been significantly improved and typically more than 500 mg PZP can be obtained from 1 liter pregnancy plasma. Furthermore, the stability of PZP at different temperatures on storage was studied. In liquid nitrogen PZP can be maintained in native dimeric form with intact thiolester for many years. The storage of native PZP with intact functional properties during and after purification is an obligatory prerequisite to elucidate the biological role of PZP. The receptor binding domain of PZP can be cleaved from the PZP-methylamine complex by papain and isolated from the other peptides by S-200 gel filtration. The cleavage site was determined and the C-terminal fragment was identified with several site-specific monoclonal antibodies against PZP.

Binding Sites↗

PubMed1996

Presence of S-100 beta in cholinergic neurones of the rat hindbrain.

The double staining of S-100 beta and choline acetyltransferase (ChAT) revealed that S-100 beta immunoreactivity was localized in most, but not in all, cholinergic neurones in the somatomotor nuclei of the cranial nerves and in the ambiguus nucleus. S-100 beta was present in almost all cholinergic neurones in the brain stem reticular, red, vestibular (excluding medial), mesencephalic trigeminal and cerebellar nuclei. However, S-100 beta immunoreactivity was lacking in cholinergic neurones in the parabrachial complex, the dorsal motor nucleus of the vagal nerve and most sensory nuclei. No S-100 beta-positive neurones lacked ChAT immunoreactivity. Taken together with the fact that the vulnerability of motoneurones to axotomy is markedly reduced in the first 3 postnatal weeks, during which period neuronal S-100 beta appears and increases, a possible effect of S-100 beta on the survival of cholinergic motoneurones may be suggested.

Animals↗

PubMed1996

The conformational state of human alpha 2-macroglobulin influences its dissociation into half-molecules by sodium thiocyanate.

Sodium thiocyanate dissociates native human alpha 2-macroglobulin into half-molecules consisting of two disulfide-bonded subunits, when the salt concentration is equal to or exceeds 1.2 M. Incubation with 1.6 M sodium thiocyanate for 1 h at 22 degrees C dissociates about 90% of alpha 2-macroglobulin into half-molecules. The half-molecules remain stable when the concentration of sodium thiocyanate is reduced to 0.2 M or zero, demonstrating that reassociation does not occur under these conditions. The internal thiol esters of the half-molecules are intact because they can be exposed by treatment with methylamine or trypsin. The noncovalent interaction between the disulfide-bonded dimers is stronger in the "closed-trap" than in the "open-trap" conformation of alpha 2-macroglobulin. The cleavage in the bait region by trypsin makes alpha 2-macroglobulin completely stable toward dissociation, and alpha 2-macroglobulin remains in a tetrameric state in 2.2 M sodium thiocyanate even when trypsin is not covalently bound to it. The increase in fluorescence with time indicates that conformational changes occur as a consequence of dissociation.

Humans↗

PubMed1996

Preparation and characterization of a C-terminal fragment of pregnancy zone protein corresponding to the receptor-binding peptide from human alpha 2-macroglobulin.

Digestion of the pregnancy zone protein with papain at pH 4.5 yields an 18 kDa C-terminal fragment. This fragment consists of the 145 C-terminal amino-acid residues cleaved at Asn-1288 Ile and is homologous to the C-terminal receptor binding fragment of human alpha 2-macroglobulin obtained by cleavage with papain. The fragment contains an intrachain disulfide bond between 1308Cys and 1423Cys corresponding to that between 1304Cys and 1419Cys in alpha 2-macroglobulin. An oligosaccharide chain, is present in the C-terminal fragment of pregnancy zone protein as in human alpha 2-macroglobulin. The PZP C-terminal fragment was demonstrated to bind to the LRP/alpha 2M-receptor. Both the pregnancy zone protein and alpha 2-macroglobulin fragments bind three mAb's (alpha 1:1, R35, and 7H11D6) generated against alpha 2-macroglobulin. The mAb 7H11D6 was generated against the alpha 2-macroglobulin-proteinase complex (Isaacs, I.J., Steiner, J.P., Roche, P.A., Pizzo, S.V. and Strickland, D.K. (1988) J. Biol. Chem. 263, 6709-6714) and the binding of this to the C-terminal fragments of both pregnancy zone protein and alpha 2-macroglobulin indicates that both proteins use the same receptor recognition site for binding to the LRP/alpha 2M-receptor.

Amino Acid Sequence↗