Biomedical subjects

F van Leuven

Publications and source records attributed to F van Leuven.

At least 19 recordsLinked to original sources

PubMed2002

Unusual association of Hodgkin's disease and sarcoidosis.

We report a 51-year-old patient who developed abdominal lymphadenopathy following Hodgkin's disease seven years after she was diagnosed as having sarcoidosis. The patient had been treated with steroids, methotrexate and azathioprine. After three cycles of chemotherapy for Hodgkin's disease, the patient again developed sarcoidosis in the mediastinal lymph nodes. A greater awareness of the co-existence of sarcoidosis and Hodgkin's disease could circumvent the diagnostic difficulties.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2000

Single and multiple transgenic mice as models for Alzheimer's disease.

Transgenic mice expressing in brain different mutant forms of the Amyloid Precursor Protein, develop functional, cognitive and pathological defects which resemble or are reminiscent of symptoms observed in Alzheimer's disease (AD) patients. The late development of amyloid plaques in aging transgenic APP mice is needed to warrant that the earlier behavioural and cognitive defects are informative for the human disorder. We describe and discuss our work, the rationale behind the approach and the techniques used to generate these APP transgenic mice, including specific experimental problems. The APP transgenic mouse models are being comprehensively characterized and offer excellent perspectives for the study and definition of early biochemical and pathological aspects that are not accessible in human AD patients. The ongoing combination by breeding with other transgenic mouse strains, i.e. mice overexpressing human Presenilin 1, ApoE 4 and protein tau to generate "multiple" transgenic mice, offer additional potential to define the pathological interactions of these genetic factors, known to be involved, directly or indirectly, in dementia of the Alzheimer type. Finally, it must be the aim to obtain transgenic mice that not only model amyloidogenesis, but also the neurofibrillary tangle pathology and the involvement of protein tau.

Alzheimer Disease↗

PubMed1999

Detection of alpha-macroglobulin in the heart of mice infected with Trypanosoma cruzi.

The aim of this work was to investigate the presence of alpha-macroglobulin (AM) in the heart of mice during acute experimental Chagas' disease and to study its localization as related to the presence of the parasite and/or to their antigens. Frozen heart tissue sections obtained from Swiss albino male mice at different days postinfection with Trypanosoma cruzi were examined for triple immunofluorescence in response to parasite antigen (green), AM (red), and nuclei (blue) from both cells. AM was found in the heart of all the infected animals studied. Parasites were seen arranged in nests inside heart muscle cells. Usually, AM staining corresponded in position to parasite nests and to their antigens spread in large areas of the myocardium. The most intense staining of AM was observed at days 9 and 11 postinfection, when the highest tissue-infection level occurs. These observations relate the presence of AM to that of T. cruzi antigen in the same areas of the inflamed myocardium of the chagasic animals.

Animals↗

PubMed1998

Elderberry (Sambucus nigra) contains truncated Neu5Ac(alpha-2,6)Gal/GalNAc-binding type 2 ribosome-inactivating proteins.

Analysis of affinity-purified preparations of the fetuin-binding proteins from elderberry bark and fruits revealed besides the previously reported Neu5Ac(alpha-2,6)Gal/GalNAc-specific type 2 ribosome-inactivating proteins (RIP) the occurrence of single chain proteins of 22 kDa, which according to their N-terminal amino acid sequence correspond to the second part of the B chain of the respective type 2 RIP. Both proteins are very similar except that the polypeptides of the fruit lectin are 10 amino acid residues longer than these from the bark lectin. Our findings not only demonstrate the occurrence of carbohydrate-binding fragments of type 2 RIP but also provide further evidence that type 2 RIP genes give rise to complex mixtures of type 2 RIP/lectins in elderberry.

Acetylgalactosamine↗

PubMed1998

Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin.

We have examined the role of Furin in postimplantation-stage mouse embryos by analyzing both the expression pattern of fur mRNA and the developmental consequences of a loss-of-function mutation at the fur locus. At early stages (day 7.5), fur mRNA is abundant in extraembryonic endoderm and mesoderm, anterior visceral endoderm, and in precardiac mesoderm. 1 day later fur is expressed throughout the heart tube and in the lateral plate mesoderm, notochordal plate and definitive gut endoderm. Embryos lacking Furin die between days 10.5 and 11.5, presumably due to hemodynamic insufficiency associated with severe ventral closure defects and the failure of the heart tube to fuse and undergo looping morphogenesis. Morphogenesis of the yolk sac vasculature is also abnormal, although blood islands and endothelial precursors form. Analysis of cardiac and endodermal marker genes shows that while both myocardial precursors and definitive endoderm cells are specified, their numbers and migratory properties are compromised. Notably, mutant embryos fail to undergo axial rotation, even though Nodal and eHand, two molecular markers of left-right asymmetry, are appropriately expressed. Overall, the present data identify Furin as an important activator of signals responsible for ventral closure and embryonic turning.

Animals↗

PubMed1997

Alpha-2-macroglobulin binds to the surface of Trypanosoma cruzi.

Trypanosoma cruzi, the causative agent of Chagas disease, infects vertebrate cells after an initial step of parasite/host-cell recognition. Alpha-2-macroglobulin (A2M), an important type of physiological proteinase inhibitor found in tissues and in the plasma of mammals, inhibits cell invasion by T. cruzi and accumulates in sites of the inflamed myocardium associated with parasite antigens. To study whether A2M would bind to T. cruzi, an indirect immunofluorescence reaction was performed using two different anti-mouse A2M sera. Intense labeling was observed in the membrane lining the cell body and the flagellum of bloodstream trypomastigotes obtained from experimentally infected mice in the peak of parasitemia, suggesting that the antisera recognize plasma A2M associated with the parasite surface. Metacyclic trypomastigotes obtained in a serum-free defined medium reacted with anti-A2M only after previous incubation with purified human A2M. Enzyme-linked immunosorbent assay (ELISA) studies were applied to characterize better the binding of native (N-A2M) and of proteinase-complexed (P-A2M) forms of A2M. The "in vitro" binding of N-A2M to trypomastigotes was better at pH 5.0, followed by pH 10.0 and pH 7.4. Cysteinly and serine proteinase inhibitors, E-64 and STI, respectively, inhibited the reaction. P-A2M also bound to T. cruzi in a dose-dependent way. Flow-cytometry studies showed that about 80% of the parasites stained with fluorescein isothiocyanate (FITC)-labeled P-A2M (50 micrograms/ml) with high affinity at pH 7.4 (but also at pH 10.0) in a process that was reverted by the addition of unlabeled P-A2M or the calcium-chelator agent EDTA and also by incubation at an acid pH (4.0). These results suggest that (a) native-A2M binds to T. cruzi proteinase(s) and (b) T. cruzi expresses a receptor(s) that binds proteinase-complexed A2M.

Animals↗

PubMed1996

One-step immunoaffinity purification and partial characterization of hypophyseal growth hormone from the African catfish, Clarias gariepinus (Burchell).

Growth hormone (GH) was purified from African catfish (Clarias gariepinus) pituitary extracts in a single step by use of immunoaffinity chromatography. A monoclonal antibody to chicken GH, which labels the catfish hypophyseal somatotropes in immunocytochemistry, was coupled to CNBr-activated Sepharose, and crude alkaline pituitary extracts were run over the immunoadsorbent. Reversed-phase high-performance liquid chromatography analysis of the eluted material suggested heterogeneity, whereas silver staining upon SDS-polyacrylamide gel electrophoresis showed one single band with an estimated molecular weight between 22,000 and 23,000 Da. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the same preparation revealed the presence of several components with molecular weights ranging from 20,170 to 20,900 Da. The amino terminus of the protein was homogeneous, and the first 50 residues matched the proposed sequence of GH from two other siluran species (Ictalurus punctatus and Pangasius pangasius), except for one substitution at position 3. These data unequivocally confirm the identity of the purified molecule as suggested by immunochemical evidence. The bioactivity of the GH preparation was demonstrated by the short-term effect of GH on T3 plasma levels in juvenile catfish.

Amino Acid Sequence↗

PubMed1995

Heterogeneity in the synthesis of alpha-macroglobulins in outbred Swiss albino mice acutely infected with Trypanosoma cruzi.

Alpha-Macroglobulins (AM) are protease inhibitors with important roles in inflammation and in immunomodulation that behave as acute-phase proteins in many experimental models. In the present work the levels of AM in the plasma of outbred Swiss albino mice acutely infected with Trypanosoma cruzi were studied. The results showed that increased levels of AM were present in the majority of the infected mice and that AM levels increased independently of the rise in parasitaemia. There was a high degree of heterogeneity in the intensity of the modulation of AM levels as well as in the kinetics of AM synthesis. This heterogeneity was related neither with the intensity of infection nor with the sex of the host. No correlation between AM levels and survival to the acute phase could be observed in the outbred mice. The consequence of such a heterogeneity is unclear, although AM as immunoregulatory molecules could play a role in the development of the symptoms of the chronic phase of Chagas' disease.

Acute Disease↗

PubMed1994

Genomic cloning of the mouse LDL receptor related protein/alpha 2-macroglobulin receptor gene.

The LDL receptor-related protein (LRP) or alpha 2-macroglobulin receptor (A2mr) is encoded by a 15-kb mRNA in mouse and human. Probes encompassing different regions of the mouse cDNA were used to isolate clones from a cosmid library of mouse strain 129. Four overlapping cosmids were used for restriction mapping and Southern blot analysis. This map and hybridization data obtained with oligonucleotide probes from the 5' and 3' ends of the Lrp cDNA demonstrated that the mouse gene is approximately 85 kb in size. The Lrp promoter region was sequenced and reveals strong evolutionary conservation of putative regulatory elements between mouse and human. The present study will facilitate detailed elucidation of the function of LRP in vivo.

Animals↗

PubMed1994

Heterogeneity in the plasma levels of two acute-phase proteins in mice from inbred strains infected with Trypanosoma cruzi.

We analyzed the variations observed in the plasma levels of both alpha-macroglobulins (AM) and serum amyloid P (SAP) in mice from three different inbred strains (C3H, Balb/C and C57black/6) acutely infected with Trypanosoma cruzi. SAP levels increased in C57black/6 and Balb/C mice but not C3H mice. AM levels increased in all C3H mice but not in C57black/6 mice and rose slightly in only 43% of the Balb/C mice. AM and SAP levels are differently modulated in patterns that may be strain-determined.

Animals↗

PubMed1993

Cloning and characterization of the lectin cDNA clones from onion, shallot and leek.

Characterization of the lectins from onion (Allium cepa), shallot (A. ascalonicum) and leek (A. porrum) has shown that these lectins differ from previously isolated Alliaceae lectins not only in their molecular structure but also in their ability to inhibit retrovirus infection of target cells. cDNA libraries constructed from poly(A)-rich RNA isolated from young shoots of onion, shallot and leek were screened for lectin cDNA clones using colony hybridization. Sequence analysis of the lectin cDNA clones from these three species revealed a high degree of sequence similarity both at the nucleotide and at the amino acid level. Apparently the onion, shallot and leek lectins are translated from mRNAs of ca. 800 nucleotides. The primary translation products are preproproteins (ca. 19 kDa) which are converted into the mature lectin polypeptides (12.5-13 kDa) after post-translational modifications. Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.

Allium↗

PubMed1993

A monoclonal antibody to the alpha 2 integrin subunit cross-reacts with RGD-dependent epitopes in fibrinogen.

Monoclonal antibodies were raised against platelet integrins purified by affinity chromatography. Two monoclonal antibodies (5C5 and 3H8) reacted with the purified alpha 2 subunit of VLA-2 in Western blotting. The monoclonal antibody 3H8 also reacted with fibrinogen in Western blots and in ELISA tests. CNBr fragmentation of the alpha chain of fibrinogen generated two peptides which were still recognized by this monoclonal antibody in Western blotting. N-terminus sequencing of these two fragments showed that they were non-overlapping fragments of the fibrinogen alpha-chain with as only common epitope an RGD(F)/RGD(S) sequence. Dot blots and ELISA tests showed that the antibody 3H8 also recognized, however with lower affinity, fibronectin and collagen IV, which are RGDS containing extracellular matrix proteins. The assumption that Mab 3H8 recognizes an RGD sequence, was further supported by the findings that the binding of Mab 3H8 to fibrinogen was partially inhibited by RGDF containing peptides and that the antibody was able to inhibit platelet aggregation.

Amino Acid Sequence↗

PubMed1992

Expression of the novel intermediate filament-associated protein restin in Hodgkin's disease and anaplastic large-cell lymphoma.

In this study, the expression of the novel intermediate filament protein Restin in human tissues was analyzed. Restin expression was studied by immunohistochemistry using polyclonal and monoclonal antibodies. Restin was not detected in normal tissues, a range of B- and T-cell non-Hodgkin's lymphomas, and nonlymphoid tumors. However, Restin was present in Reed-Sternberg cells and variants thereof in Hodgkin's disease, with the exception of the lymphocyte-predominant, paragranuloma subtype. Restin was also highly expressed in anaplastic large-cell lymphoma (so-called Ki-1 lymphoma). As expected, Restin was also expressed in Hodgkin cell lines L428, L428KSA, Co, and KM-H2 and the anaplastic large-cell lymphoma cell line Karpas 299, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, as well as Northern blotting. The presence of Restin in both Hodgkin's disease and anaplastic large-cell lymphoma is intriguing and might indicate a role of this structural protein in the pathogenesis of both conditions.

Antibodies, Monoclonal↗

PubMed1992

The primary sequence and the subunit structure of mouse alpha-2-macroglobulin, deduced from protein sequencing of the isolated subunits and from molecular cloning of the cDNA.

Mouse plasma alpha-2-macroglobulin (m alpha 2M) was isolated and the N-terminal amino-acid sequences determined after separation of the 165-kDa and 35-kDa subunits. These sequences were compared to the protein sequence predicted by the cDNA, which was cloned from a mouse liver library and sequenced. From these data it is evident that both subunits are encoded by one mRNA of approximately 5 kb expressed predominantly in liver. The smaller subunit, with the N-terminal sequence DLSSSDLT, comprises the C-terminal 257 residues of m alpha 2M and is derived from a single-chain precursor probably by proteolytic processing at an arginine residue in the sequence PTRDLSS. Analysis of the predicted protein further showed all the salient features of a proteinase inhibitor of the macroglobulin family: a bait region that deviates from all known sequences in this family, a very conserved internal thiolester site and conserved cysteine residues and putative N-glycosylation sites. The synthesis of m alpha 2M in adult liver was demonstrated by Northern blotting and in fetal liver by in-situ hybridization. Transient transfection of COS cells with the cDNA under control of a viral promoter demonstrated the secretion and partial processing of m alpha 2M in the culture medium. In plasma the level of m alpha 2M was found to be stable as expected for the murine counterpart of human plasma alpha-2-macroglobulin. The possibilities of using the mouse as a genetic model to study this proteinase inhibitor in vivo are discussed.

Amino Acid Sequence↗

PubMed1992

The closely related homomeric and heterodimeric mannose-binding lectins from garlic are encoded by one-domain and two-domain lectin genes, respectively.

Lectin cDNA clones for two different lectins from garlic (Allium sativum L.) bulbs, ASAI and ASAII (ASA, Allium sativum agglutinin), were isolated and characterized. The first lectin, ASAI, is a heterodimer composed of two different subunits of 11.5 kDa and 12.5 kDa. It is translated from an mRNA of 1400 nucleotides encoding a polypeptide of 306 amino acids with two very similar domains. N-terminal sequencing of the two polypeptides of the mature lectin confirmed that both subunits are derived from the same precursor and that each corresponds to one of the two domains in the sequence. In contrast to ASAI, the second garlic lectin, ASAII, is a homodimer of two identical 12-kDa subunits. It is translated from an mRNA of approximately 800 nucleotides encoding a polypeptide of 154 amino acids. Interestingly, the coding region of the ASAII cDNA clones is almost identical to that of the second domain of the ASAI cDNA clones.

Amino Acid Sequence↗

PubMed1989

A cluster of alpha 2-macroglobulin-related genes (alpha 2 M) on human chromosome 12p: cloning of the pregnancy-zone protein gene and an alpha 2M pseudogene.

The characterization of two alpha 2-macroglobulin (alpha 2M)-related genomic clones, isolated from two human genomic libraries by use of alpha 2M cDNA [Kan et al., Proc. Natl. Acad. Sci. USA 82 (1985) 2282-2286] as a probe, is reported. Sequence comparison of the clone EPZP6 with the human alpha 2M cDNA revealed the presence of five exons with the proper splice signals. Alignment of the corresponding amino acid (aa) sequence of these exons with the published partial pregnancy-zone protein (PZP) aa sequence (Sottrup-Jensen et al., Proc. Natl. Acad. Sci. USA 81 (1984) 7353-7357] showed a perfect match, thereby identifying EPZP6 as a PZP genomic clone. The clone MPAM16 showed a considerable degree of sequence conservation when compared to the human alpha 2M cDNA sequence, and several putative exons were identified. However, a frame-shift mutation leading to a premature stop codon was found in the coding sequence, classifying this gene as an alpha 2M pseudogene. Human alpha 2M, PZP and the related pseudogene were mapped to the human chromosome 12p12-13, with the help of gene-specific probes and in situ hybridization. This result was confirmed in Southern-blot experiments with DNA from a human-Ltk- mouse somatic-cell hybrid containing only a human isochromosome 12p in a mouse background.

Amino Acid Sequence↗

PubMed1988

Post-translational modification of the beta-subunit of the human fibronectin receptor.

Monoclonal antibody DH12, directed against the beta-subunit of the fibronectin receptor recognizes a doublet of proteins (100 and 110 kDa) in Western blots of solubilized whole fibroblasts. Pulse-chase experiments with [35S]methionine in human skin fibroblasts suggested that the two proteins might be metabolically related as precursor (100 kDa) and product (110 kDa). Endo H digestion and [3H]fucose labeling suggested that maturation converted the high-mannose oligosaccharides (100 kDa) to the endoglycosidase H resistant complex type (110 kDa). This was supported by N-glycanase digestion and by chemical deglycosylation which showed a single polypeptide. Surface iodination of intact cells labeled only the presumed mature beta-subunit.

Antibodies, Monoclonal↗

PubMed1984

Human alpha 2 macroglobulin.

Human alpha 2 macroglobulin combines two unique features: the non-active site directed inhibition of virtually all endoproteases and the selective clearance of alpha 2 M-endoprotease complexes by receptor-mediated endocytosis. To study the molecular details of the mechanisms involved, primary amines were found to be worthwhile probes at three specific levels: the inactivation of native alpha 2 M, the derivatization by factor XIII and the cellular process of receptor-recycling. In this paper published data are supplemented with recently obtained evidence to discuss and speculate on the possible action or involvement of transglutaminase activities, indicated by the effects of the primary amines.

Acyltransferases↗