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Biomedical subjects

A Van de Voorde

Publications and source records attributed to A Van de Voorde.

At least 19 recordsLinked to original sources
PubMed1995

Transmembrane segments critical for potassium channel function.

The relative contribution of the transmembrane segments in the alpha-subunit of Shaker-type potassium channels was investigated in relation to potassium channel function. Starting from a wild-type Kv1.1 channel, four different deletion mutants were made, missing respectively transmembrane segments S1 and S2, S2 and S3, S1 to S3, and S1 to S4. To ensure the assembly of the different subunits, the hydrophylic N-terminal domain was always conserved. The lack of transmembrane segments S1 to S4 converts a depolarization-activated WT Kv1.1 channel with outward rectification into a hyperpolarization-activated channel with inward rectification. In contrast, mutant channels missing transmembrane segments S1 and S2, S2 and S3, or S1 to S3 did not reveal functional expression.

Animals↗
PubMed1994

The phosphorylation state of tau in the developing rat brain is regulated by phosphoprotein phosphatases.

The paired helical filaments (PHFs) in Alzheimer's disease neurofibrillary tangles are composed of PHF-tau which is thought to be hyperphosphorylated because several residues in postmortem samples of PHF-tau and human fetal tau are phosphorylated while the corresponding sites are not phosphorylated in autopsy-derived normal adult human brain tau. To determine how the phosphorylation of these sites is regulated, we isolated tau from rat brains at different embryonic and postnatal ages in the presence of okadaic acid to obtain tau in its most native in situ phosphorylation state. Fetal tau was highly phosphorylated from embryonic day 18 (E18) until postnatal day 11 (P11). Thereafter, the levels of fetal tau diminished as did its phosphorylation state concomitant with the appearance of the five adult tau isoforms. Several phosphorylation-dependent antibodies (i.e. AT270, AT8, AT180, T3P, and PHF1) that recognize PHF-tau also recognized these tau isoforms, albeit at reduced levels in the mature rat brain. This suggests that Thr172, Ser193, Thr222, Ser387, and Ser395 are normal sites of phosphorylation in rat brain tau. The inclusion of OK in the microtubule assembly buffers did not alter the ability of tau to bind microtubules at any age. However, phosphatases were activated and kinases were down-regulated in the rat brain after P12 since adult tau proteins were partially dephosphorylated at and beyond this time in the absence of OK. Protein phosphatase 2A (PP2A) and 2B (PP2B) activities in the adult rat brain extracts dephosphorylated tau efficiently, but protein phosphatases in extracts of the P6 rat brain did not have a similar effect. This suggests that the sensitivity of tau to OK after P12 may be regulated by the de novo induction of adult brain phosphatases. Finally, PP2A and/or PP2B in adult rat brain extracts dephosphorylated tau in a site-specific manner. Thus, PP2A and PP2B (or closely related phosphatases) may regulate the phosphorylation state of adult tau isoforms in vivo, and the generation of PHF-tau in the AD brain may result from the abnormal inactivation of similar phosphatases.

Animals↗
PubMed1994

Characterization of a rat C6 glioma-secreted follistatin-related protein (FRP). Cloning and sequence of the human homologue.

A protein was isolated from rat C6 glioma-conditioned medium and was biochemically characterized. The heparin-binding protein has a native molecular mass of 55-75,000 Da, a molecular mass of 40-48,000 Da under denaturing conditions, and a pI of 5.0-6.0. Based on the determined partial amino acid sequences, the full lenght cDNA encoding the rat and human proteins were cloned. The cDNA sequences identified the isolated rat and human protein as the homologue of a recently reported mouse osteoblast-transforming-growth-factor-beta 1-inducible protein, encoded by the TSC-36 gene [Shibanuma, M., Mashimo, J., Mita, A., Kuroki, T. & Nose, K. (1993) Eur. J. Biochem. 217, 13-19]. Analysis of the human, rat and mouse amino acid sequences indicates that these proteins are highly conserved (> 92% sequence identity). Sequence similarities with follistatin and the follistatin-like domain of agrin are revealed. The relationship with follistatin and agrin points to possible common functions for the cloned follistatin-related proteins (FRP). The protein has no effect on the inhibitory action of transforming growth factor-beta 1, on CCl-64 cell growth.

Agrin↗
PubMed1993

Identification by microsequencing of lipopolysaccharide-induced proteins secreted by mouse macrophages.

The conditioned medium of the murine macrophage PU5.1.8 was analyzed by two-dimensional gel electrophoresis in order to detect LPS-induced proteins. Spots of interest were identified by microsequencing of internal peptides generated by limited in situ acid hydrolysis. In total conditioned medium, several monokines (TNF-alpha and macrophage inflammatory protein-1 alpha and 1 beta) were identified as LPS-induced spots. Because minor spots could be masked by the complexity of the 2-D pattern, conditioned medium was successively fractionated by zinc precipitation and affinity chromatography (Procion red and Con A agarose). Zinc supernatant fraction, Procion red flow-through, and Con A eluate fractions were further analyzed by 2-D gel electrophoresis for the presence of LPS-induced spots. In these fractions serum amyloid A3, lipocalin 24p3, cathepsin B, and plasminogen activator inhibitor-I were characterized as LPS-induced proteins secreted by macrophages. Lipocalin 24p3 protein was retrieved for the first time. In addition to these proteins that follow a classical secretory pathway, several cellular proteins (mainly ribosomal proteins) were retrieved as LPS-induced proteins in the conditioned medium. Control experiments argue against the obvious explanation that the latter observation is caused solely by cellular leakage.

Amino Acid Sequence↗
PubMed1992

Production of functionally active murine and murine::human chimeric F(ab')2 fragments in COS-1 cells.

We report here the synthesis, in nonlymphoid cells, of two functionally active recombinant F(ab')2 fragments directed against the tumor marker, human placental alkaline phosphatase (hPLAP). The truncated heavy chain (HC) sequences, E6Hf2 and E6Hy3f2, of the murine F(ab')2 fragment, E6F2, and of the murine::human chimeric F(ab')2 fragment, E6(Hy3,kappa)F2, respectively, were engineered by introducing an in-phase stop codon within the second constant domain of the corresponding parental HC sequence. The antibody-encoding genes were placed under control of the simian virus 40 late promoter and each HC sequence, together with the light chain (LC) sequence, was transiently expressed in COS-1 cells. The truncated HCs were correctly synthesized, processed and assembled with the murine LC and subsequently secreted into the culture medium as functionally active entities with stable hinge region interactions. These results indicate that, under the conditions used, the hinge region was sufficient for the formation of divalent molecules. However, Western blotting revealed the presence of hPLAP-binding half-molecules of E6F2, which was not the case for E6(Hy3,kappa)F2. Since E6F2 and E6(Hy3,kappa)F2 mainly differ by the length of their hinge region (22 and 62 aa residues, respectively) and the number of inter-HC disulfide bridges (four and eleven, respectively), it may be concluded that F(ab')2 fragments with an extended hinge region and several inter-HC disulfide bridges are formed more efficiently.

Alkaline Phosphatase↗
PubMed1992

Monoclonal antibodies with selective specificity for Alzheimer Tau are directed against phosphatase-sensitive epitopes.

A modified form of the microtubule-associated protein Tau is the major component of the paired helical filaments (PHF) found in Alzheimer's disease. The characterization of these posttranslational Tau modifications is hindered by the lack of sufficient PHF-Tau-specific markers. Here we describe several monoclonal antibodies, prepared by immunization with PHF, two of which showed a selective specificity for PHF-Tau without cross-reactivity with normal Tau. Epitope recognition by these two monoclonals was sensitive to alkaline phosphatase treatment. In Western blotting these monoclonal antibodies reacted specifically with the abnormally phosphorylated epitopes on Alzheimer's disease-associated PHF-Tau. One of the new antibodies can be used for the construction of a sandwich enzyme-linked immunosorbent assay for the specific detection of PHF-Tau without cross-reactivity to normal Tau proteins.

Animals↗
PubMed1992

Specific monoclonal antibodies against normal microtubule-associated protein-2 (MAP2) epitopes present in Alzheimer pathological structures do not recognize paired helical filaments.

We have developed monoclonal antibodies that detect normal microtubule-associated protein-2 (MAP2) epitopes in routinely fixed, paraffin-embedded tissue. The somatodendritic distribution of MAP2 in bovine and human nervous tissue was confirmed with several of these antibodies. Furthermore, some of these antibodies immunohistochemically labeled certain pathological structures in Alzheimer brain, especially neurites in senile plaques. Electron microscopic observations, however, indicate that these MAP2 epitopes are not located in the Alzheimer paired helical filaments themselves, but in amorphous granular structures coexistent with them. While the pathological nature of these structures is undetermined, they may represent artefactual modifications of normal cytoskeletal components.

Alzheimer Disease↗
PubMed1992

Affinity purification of human tau proteins and the construction of a sensitive sandwich enzyme-linked immunosorbent assay for human tau detection.

Immunoaffinity chromatography with a monoclonal antibody produced against bovine tau protein was used to purify tau proteins from human brain. Fifty grams of brain tissue yielded approximately 2 mg of pure tau proteins. The affinity-purified human tau was used to produce a high-titered rabbit anti-human tau serum. The monoclonal anti-tau antibody and the polyclonal rabbit anti-tau serum were then used to construct a sandwich enzyme-linked immunosorbent assay for detection of human tau proteins, with a sensitivity of 1 ng/ml.

Animals↗
PubMed1992

Magnetic beads in suspension enable a rapid and sensitive immunodetection of human placental alkaline phosphatase.

The kinetics and efficiency of the interaction between placental alkaline phosphatase and a monoclonal antibody (laboratory number 327) were determined by immunoassay using microtitre plates or magnetic beads. While only up to 45% of placental alkaline phosphatase was bound to microwells precoated with this antibody, even after prolonged incubation, no less than 60% and 100% binding were reached using magnetic beads after 1 and 3 h incubations, respectively. High-molecular-mass placental alkaline phosphatase and complexed placental alkaline phosphatase forms were also completely bound to magnetic beads in the presence of deoxycholate (up to 9 g/l for serum samples). The assay sensitivity was improved up to 4-fold. In addition, 100% binding of the antigen was achieved during simultaneous incubation of magnetic beads, monoclonal antibody (125 micrograms/l), and placental alkaline phosphatase. This one-step enzymatic assay, based on magnetic beads, is an attractive alternative to the classic assay performed in microtitre plates, enabling rapid, precise, and sensitive antigen detection, and only necessitating a minimum of laboratory equipment.

Alkaline Phosphatase↗
PubMed1991

Isolation of IgG1-secreting switch variants from IgM hybridomas produced after in vitro immunization.

IgG1-secreting variants have been isolated from three different IgM-secreting hybridomas, in two instances following in vitro immunization. The method used was based on sequential sublining in combination with selection by an IgG1-specific two-site ELISA system employing two different IgG1-specific polyclonal antisera. Idiotypic identity between the IgG1 variants and their respective IgM parent was demonstrated using syngeneic anti-idiotypic antisera. The antigen binding specificity in the IgG1 variants was also conserved. Isolation of naturally occurring IgG1 switch variants from IgM-secreting hybridomas that are produced after in vivo immunization offers a solution to the major disadvantages associated with the generation of IgM hybridomas.

Alzheimer Disease↗
PubMed1988

Expression in non-lymphoid cells of mouse recombinant immunoglobulin directed against the tumour marker human placental alkaline phosphatase.

From a mouse hybridoma cell line secreting a monoclonal antibody directed against the tumour marker human placental alkaline phosphatase, mRNA coding for the H and L chains of this antibody was isolated and cloned as cDNA. Sequence analysis of the H and L chain cDNAs confirmed the IgG2b,kappa subtype previously established. Recloning the H and L chain cDNA information into SV40-based vectors enabled us to obtain expression of functional immunoglobulin upon cotransfection into COS or CHO dhfr- cells. This illustrates that non-lymphoid cells also have the capacity to assemble active immunoglobulins.

Alkaline Phosphatase↗
PubMed1988

Expression of functional mouse antibodies directed against the tumour marker human placental alkaline phosphatase in non-lymphoid cells.

A mouse hybridoma cell line was isolated which produces monoclonal antibodies (MAbs) of the IgG2b, kappa subtype directed against the tumour-associated marker human placental alkaline phosphatase (hPLAP). The mRNAs coding for the heavy (H) and light (L) chains were cloned as cDNA copies. These genes were then separately inserted into the eukaryotic expression vector pSV23p, under control of the SV40 early promoter. Both genes were introduced with the DEAE-dextran technique in COSI cells, and 72 hr after transfection, 10 ng/ml functional antibodies could be detected in the supernatant of the cells. Permanent CHO cell lines secreting 100 ng/ml functional antibodies were established upon transfection of CHO (dhfr-) cells with the plasmids containing the H and L cDNAs and the plasmid pAdD26SVp-(A)-3 carrying the mouse dihydrofolate reductase (dhfr) gene. A plasmid construction in which we inserted a stop codon-containing sequence behind the hinge region of the H-chain cDNA sequence yielded immuno-competent F(ab')2 molecules upon transfection of COS or CHO cells. Our results indicate that not only lymphoid cells, but also non-lymphoid cells, are capable of synthesis and assembly of immunoglobulin chains that are immunologically fully competent.

Alkaline Phosphatase↗
PubMed1985

Screening of sera and tumor extracts of cancer patients using a monoclonal antibody directed against human placental alkaline phosphatase.

A sensitive endogenous enzyme immunoassay involving an anti-human placental alkaline phosphatase (PLAP) monoclonal antibody was used in the screening of sera and tumor extracts of patients with various types of cancer. In sera of breast cancer patients an incidence of 5.2% was recorded. This value rose to 43% when tumor extracts were analyzed. For lung and bronchial cancers we found 11.2% seropositive patients. On several occasions a good correlation was observed between the PLAP determinations and the histopathological staging of tumor tissue.

Adult↗
PubMed1985

Enzyme-antigen immunoassay for human placental alkaline phosphatase in serum and tissue extracts, and its application as a tumor marker.

In this enzyme-antigen immunoassay for human placental alkaline phosphatase (hPLAP; EC 3.1.3.1.) in serum and tissue extracts, polyclonal rabbit antiserum to mouse IgG2b is adsorbed to the wells of a microtiter plate, its excess binding sites are blocked, then it is incubated with murine monoclonal anti-hPLAP and mixed with serially diluted standard or sample antigen. The amount of antigen bound is determined by measuring its enzymic activity. The standard curve is linear for hPLAP concentrations of 0.2 to 1 U/L. The mean within-assay CV was 3.8% (SD 0.9%) for a serum sample and 6.1% (SD 3.0%) for a tissue extract. The respective mean between-assay CVs were: 6.7% (SD 2.0%), and 7.0% (SD 2.0%). Serum hPLAP concentrations, determined in four different dilutions, had a CV of 5.5%. We evaluated the method by standard additions and by comparing dilution curves for purified hPLAP, hPLAP in serum, and hPLAP in tissue extracts. The upper limit of activity in normal subjects was 0.1 U/L for serum samples, and 1.0 mU/g wet weight of tissue for tissue extracts. hPLAP activity was increased in 9.8% of all cancer patients, and in 40% of ovarian cancer patients. Almost half of the tumor biopsies were positive for hPLAP activity, and 94% of the biopsies from ovarian neoplasia had an increased activity of this isoenzyme. Of the nonmalignant tissues examined, normal lung tissue had the highest hPLAP activity.

Alkaline Phosphatase↗
PubMed1985

Human placental alkaline phosphatase in benign and malignant ovarian neoplasia.

In benign and malignant ovarian tumor patients, human placental alkaline phosphatase (HPLAP) was determined in serum and extracts from surgical tumor biopsies using a highly specific enzyme-antigen immunoassay based on a mouse monoclonal antibody (E6) to HPLAP. Serum HPLAP levels greater than or equal to 0.1 unit/liter were found in 58% of ovarian cancer patients. Serum carcinoembryonic antigen levels were positive (greater than 5.4 ng/ml) in 17% of these patients. HPLAP was detected in extracts from 13 of the 14 tumors investigated (range, 2.4 to 557 milliunits/g). Only the mixed heterologous Müllerian sarcoma was negative. The highest HPLAP content of normal ovarian tissue was 1.1 milliunits/g. The amount of heat-stable and L-p-bromotetramisole-insensitive alkaline phosphatase was in all cases much higher than the fraction recognized by E6. The neoplastic origin of HPLAP was confirmed immunohistochemically on paraffin sections by an indirect avidin-biotin-peroxidase staining procedure using E6. The staining pattern was compared to the histochemical distribution of total alkaline phosphatase on adjacent sections. A consistency was found between the amount of HPLAP in tissue extracts and its immunohistochemical distribution. In all the tumors, staining for HPLAP was observed mainly on the plasma membranes of carcinoma cells. In 9 of the 10 carcinomas, the histological distribution of HPLAP and also of total alkaline phosphatase was heterogeneous. HPLAP staining, present in one of five normal ovaries, was restricted to germinal inclusion cysts. The present results support the hypothesis that serous ovarian tumors originate from these cysts.

Alkaline Phosphatase↗
PubMed1983

A new restriction endonuclease from Acetobacter pasteurianus.

A restriction endonuclease, ApaI, has been partially purified from Acetobacter pasteurianus. This enzyme cleaves bacteriophage lambda DNA and Simian virus 40 DNA at one site, adenovirus-2 DNA at more than nine sites, but it does not cleave phi X174 DNA nor plasmid pBR322 DNA. This enzyme recognizes the sequence (formula; see text) and cuts at the sites indicated by the arrows.

Acetobacter↗