Biomedical subjects

V Erfle

Publications and source records attributed to V Erfle.

At least 91 records · Page 5Linked to original sources

PubMed1991

Isolation of a cathepsin B-encoding cDNA from murine osteogenic cells.

Cathepsin B-encoding cDNA (CTSB) clones have been isolated from a lambda gt10 library of a murine osteosarcoma by differential screening during a search for genes which are typically expressed during osteogenic differentiation in mouse mandibular condyles in vitro. Sequencing of the CTSB 3' end revealed that the isolated sequence contained an 825-bp 3'-noncoding region, the polyadenylation signal and the poly(A) tail. The enhanced CTSB expression during the early stages of the enchondral ossification-like process in mandibular condyles in vitro suggests that CTSB participates in the degradation of cartilage matrix prior to the synthesis of bone matrix proteins.

Animals↗

PubMed1991

Bone tumor induction after incorporation of short-lived radionuclides.

Temporally-limited internal irradiation after incorporation of short-lived bone-seeking radionuclides is a useful experimental tool for the investigation of extrinsic and intrinsic factors which modify the dose dependence of bone tumor risk. Here we describe some of the results obtained in experiments with female mice (mainly NMRI). The future aim of such experiments should be the prediction of risk of late effects using early molecular-biological changes. Molecular-biological descriptions in our model are at present very limited.

Animals↗

PubMed1991

Immunological study of the nef protein from HIV-1 by polyclonal and monoclonal antibodies.

We constructed and expressed different overlapping fusion proteins with the nef gene of HIV-1 and generated specific polyclonal rabbit and monoclonal mouse antibodies against these recombinant proteins. The rabbit antisera, one of the monoclonal antibodies as well as a serum from a HIV-1 infected patient recognized the nef protein with Mr 27 kDa in latently HIV-1 infected glioma cells in the immunoblot. In contrast, these antibodies could not detect nef in productively HIV-1 infected Molt-3 cells neither in immunoblot nor in indirect immunofluorescence assays. These results indicate the possible participation of nef in viral latency. The recombinant nef proteins were used as probes for anti-nef antibodies in human sera. We observed in 17 of 57 sera tested specific anti-nef antibodies. All of these anti-nef positive sera also contained antibodies directed against viral structural proteins. The NH2-terminal region of the recombinant nef was shown to be the major immunodominant antigenic site in the immunoblot assay.

Animals↗

PubMed1991

Serological markers as prognostic criteria for the course of HIV infection.

576 serum samples from 139 HIV infected patients were analyzed for the presence of HIV antigens, as well as anti-core, anti-env and neutralizing antibodies. The results were correlated with the clinical and immunological stages of the patients. While almost all sera were positive for anti-env antibodies, only two thirds of the same sera had antibodies to core proteins. The average antibody titres, particularly of anti-core antibodies, decreased with the onset of clinical symptoms. The presence of p24 antigen could be demonstrated in only 16% of the sera. A positive reaction for core antigen seemed to be correlated with the absence of anti-core antibodies. Env specific neutralizing antibodies were found to be present in all of the sera analyzed in the LC5-test with a maximum neutralizing capacity observed in sera from WR 4 patients. A useful serological marker must fulfil two criteria, namely positive reactivity in the majority of sera examined and a broad range of antibody titres. None of the above described parameters meet these demands. Here we describe the search for new antibody markers, for example antibodies directed against HIV regulatory proteins.

AIDS-Related Complex↗

PubMed1991

Characterization of N-myristoyl transferase inhibitors and their effect on HIV release.

Acylation of virus proteins is an important covalent modification which has been shown, in many cases, to be necessary for their normal function. Furthermore, it has been shown that cerulenin, an inhibitor of this process, inhibits formation of vesicular stomatitis virus and Rous sarcoma virus in infected cultures, as well as acylation of HIV proteins. However, in agreement with earlier reports, we found that the acylating enzyme, N-myristoyl transferase, was unaffected by cerulenin which did, however, inhibit protein synthesis, thereby making interpretation of its effects difficult. Analogues of myristic acid were found to inhibit acylation in intact cells without toxic effects on protein synthesis or mitochondrial function. Myristic acid analogues were also shown by an in vitro assay to act directly on the acylating activity (N-myristoyl transferase). Furthermore, myristic acid analogues were found to inhibit HIV release from HIV-infected cells and glucosamine, which has recently been shown to be a non-competitive inhibitor of N-myristoyl-transferase, also inhibited HIV release.

Acylation↗

PubMed1991

HIV-1 Nef protein exhibits structural and functional similarity to scorpion peptides interacting with K+ channels.

The persistent infection of human glial cells with HIV-1 is characterized by prominent expression of the Nef protein. In order to evaluate the possible role of Nef in the development of HIV-1-associated neurological disorders, we compared Nef with known neuroactive proteins. We found that HIV Nef shares sequence and structural features with scorpion peptides known to interact with K+ channels. Sequence similarity encompasses two distinct regions of scorpion peptides. Based on crystallography data, both regions in scorpion peptides cooperate in forming a common domain stabilized by ion pairs between charged amino-acid residues. Recombinant Nef protein, as well as a synthetic part of a scorpion channel active peptide (M10), reversibly increased the total K+ current of chick dorsal root ganglions in patch-clamp experiments without killing the cells. These results indicate that a region conserved in HIV Nef and scorpion peptides concurs in both structure and electrophysiological activity and suggest that Nef, like scorpion peptides, may affect neuronal cell function.

Amino Acid Sequence↗

PubMed1991

A mammary-specific promoter directs expression of growth hormone not only to the mammary gland, but also to Bergman glia cells in transgenic mice.

The whey acidic protein (WAP) promoter has been previously used to target the expression of heterologous genes to the mammary glands of transgenic mice. To direct the expression of human GH (hGH) to mouse mammary glands, hGH-coding sequences have been coupled to WAP promoter sequences (WAP-hGH). Female transgenic mice carrying the WAP-hGH constructs show expression of hGH in the mammary gland, demonstrating the functionality of the transgenes. However, when other organs from these transgenic mice were examined, high level expression of hGH was unexpectedly observed in the brains of all male and female mice. Using in situ hybridization or immunohistochemistry, hGH expression from the transgene was seen to occur specifically in Bergman glia cells. In contrast, mice carrying hGH-coding sequences linked to the metallothionein promoter do not express hGH in these cells. Neither the endogenous WAP gene nor at least three other transgenes in which heterologous genes have been placed under the transcriptional control of the WAP promoter are expressed in the brain. Thus, we propose that the combination of the WAP promoter and the hGH structural gene results in a novel tissue specificity in the Bergman glia.

Animals↗

PubMed1991

[Unexpected transgene expression of a mammary-specific growth hormone gene construct in Bergmann glial cells of the mouse].

Pronuclear microinjection was used to produce transgenic mice harboring gene constructs, in which 110 base pairs (WAP1) or 2.4 kilobases (WAP2) of the 5' flanking sequences of the whey acidic protein (WAP) gene were fused to human growth hormone (hGH)-coding sequences. Female WAP-hGH transgenic mice expressed the transgenes in the mammary gland, the expression of the WAP2-hGH transgene mirroring that of the endogenous WAP gene. When other organs were examined, high level expression of hGH was observed in the brains of WAP2-hGH transgenic mice. Using in situ hybridization and immunohistochemistry, hGH expression from the transgene was seen to occur specifically in Bergmann glia cells. While normally neither WAP nor hGH is expressed in this type of cell, it appears that a combination of the regulatory region of the WAP gene and the hGH structural gene results in a novel tissue specificity in Bergmann glia cells.

Animals↗

PubMed1990

Quantitative determination of retrovirus-specific immune complexes.

ELISA methodology was adapted to measure antigen-specific immune complexes (ASIC) containing retroviral antigens. Using ICs artificially generated from MuLV gp70 and monoclonal antibodies against gp70 we showed that ICs can be quantified by measuring the levels of antibody and antigen and comparing these to the total content of ICs. Furthermore, this method permits determination of the composition of ICs containing an excess of antigen or antibody (i.e., small or large complexes). The levels of the ASIC were correlated with the levels of ICs containing undefined antigen components as determined by the C1q ELISA. Using this method it was possible to determine levels of MuLV gp70 specific ICs in various mouse sera.

Aging↗

PubMed1990

Endogenous retroviral elements in human DNA.

Endogenous retroviruses and retroviral elements represent a substantial component of vertebrate genomes. They are inherited as stable Mendelian genes and may be activated spontaneously or by physical or chemical agents. In the human genome various retroviral elements have been detected by their relationship with mammalian endogenous and exogenous retroviruses. The structure of these elements resembles either full-length or truncated proviruses. The biological function of human retrovirus-related sequences is still unknown, but like other transposable elements, they may have contributed in shaping the eukaryotic genome. Furthermore, they exhibit a number of features giving them a potential for involvement in carcinogenesis. Expression of endogenous retroviral elements has been detected in various human tissues and cell lines and in some cases appears to be associated with human neoplasias.

DNA, Viral↗

PubMed1990

Amplification and rearrangement of c-myc in radiation-induced murine osteosarcomas.

Fifty-one radiation-induced murine osteosarcomas were investigated for alterations in c-myc gene structure and c-myc expression. Amplification of c-myc was found in 30% of BALB/c tumors and 13% of NMRI tumors. A region of common proviral integration, Mlvi-1, localized on the same region on chromosome 15, was amplified concomitantly. Multiple copies of both loci were localized on double minutes. Three of the tumors with c-myc amplification also showed rearrangements of the c-myc gene region. One of these rearrangements included the 5' and 3'-flanking sequences and the noncoding part of the third exon. Repetitive sequences were found in the 5' region of the c-myc gene, and the 3' flanking region was substituted by sequences normally present in a more distant part of chromosome 15. Increased levels of c-myc transcripts of apparently normal size were found in tumors carrying amplified c-myc sequences. Abnormally high expression of c-myc in some tumors was correlated with an early stage of osteogenic differentiation, suggesting the involvement of the c-myc gene in the control of the osteogenic differentiation of transformed cells.

Animals↗

PubMed1990

Bone formation by osteoblast-like cells in a three-dimensional cell culture.

Cells of the clonal osteogenic cell line MC3T3-E1 were seeded onto a three-dimensional matrix of denatured collagen type 1 and cultured for a period of up to 8 weeks. Specimens were analyzed by histological, enzyme histochemical, immunocytochemical, and ultrastructural methods and by in situ hybridization between day 7 and day 56 after seeding. In 56-day cultures, the MC3T3-E1 cells were arranged in a three-dimensional network and formation of bone-like tissue was indicated by calcification of a newly synthesized collagen type I matrix resembling osteoid and surrounding osteocyte-like cells. The differentiating culture showed high expression of osteocalcin and alkaline phosphatase activity. NIH3T3 fibroblasts used as control cells passed through the network of the substrate forming a confluent monolayer underneath. This culture system offers a potentially powerful model for bone formation in vitro and for investigating the osteogenic potential of bone-derived cells.

Alkaline Phosphatase↗

PubMed1990

S71 is a phylogenetically distinct human endogenous retroviral element with structural and sequence homology to simian sarcoma virus (SSV).

Human endogenous retroviral element S71 had previously been shown to contain gag- and pol-related regions and a 3' LTR-like sequence. The nucleotide sequence of S71 was determined and compared with the corresponding regions of SSV and its helper virus SSAV. The 1.48-kb S71 gag region consists of matrix protein p15 (MA)-, capsid protein p30 (CA)-, and nucleocapsid protein p10 (NC)-related sections and the 1.82-kb pol region of tether, RNase H (RH), and endonuclease/integrase (IN) sections. The S71 nucleotide sequence contains a 167 amino acid open reading frame encompassing MA. The boundaries of the S71 element are delimited by direct repeats and the entire element is 5.4 kb long. Similarity between S71 and the v-sis-bearing, defective SSV provirus also covers overall structural organization, including the presence of presumably nonretroviral sequences. Both the gag and the pol regions of S71 contain sequences highly conserved in numerous retroviruses. Phylogenetic analysis with conserved CA, RH, and IN sequences showed that of all other (C-type) human retroviral elements available for comparison, S71 is most closely related to infectious primate and murine retroviruses. This suggests that S71 represents a phylogenetic subgroup of its own. In addition we identified short ranges of conserved amino acid sequences within C-type retroviral gag and pol genes sufficient for phylogenetic analysis. Use of these may facilitate large-scale phylogenetic evaluation of C-type retroviral elements and allow rapid classification of new elements.

Amino Acid Sequence↗

PubMed1990

Pathogenicity of BALB/c-derived N-tropic murine leukemia viruses.

N-tropic murine leukemia viruses have been observed in connection with radiation-induced osteosarcomagenesis in BALB/c mice. We have investigated the bone disease-inducing potential of molecularly cloned, BALB/c-derived N-tropic viruses in the random-bred NMRI mouse strain. The germ-line virus and an exogenous virus isolate were found to induce high incidences of osteopetrosis and lymphomas and a lower incidence of osteomas. Two viruses derived from somatically acquired proviruses of independent radiation-induced osteosarcomas induced lower incidence of osteopetrosis and lymphomas. Nucleotide sequence analysis of the long terminal repeat regions and RNase T1 fingerprint analysis revealed only few differences between the isolates. The possible involvement of N-tropic murine leukemia viruses in radiation-induced osteosarcomagenesis in the BALB/c mouse strain is discussed.

Animals↗

PubMed1990

Gene expression during osteogenic differentiation in mandibular condyles in vitro.

The cartilagenous tissue of mandibular condyles of newborn mice contains progenitor cells as well as young and mature chondrogenic cells. During in vitro cultivation of the tissue, progenitor cells undergo osteogenic differentiation and form new bone (Silbermann, M., D. Lewinson, H. Gonen, M. A. Lizarbe, and K. von der Mark. 1983. Anat. Rec. 206:373-383). We have studied the expression of genes that typify osteogenic differentiation in mandibular condyles during in vitro cultivation. RNAs of the genes for collagen type I, osteonectin, alkaline phosphatase, and bone gla protein were sequentially expressed in progenitor cells and hypertrophic chondrocytes during culture. Osteopontin expression peaked in both the early and the late phase of the differentiation process. The data indicate a distinct sequence of expression of osteoblast-specific genes during osteogenic differentiation and new bone formation in mandibular condyles.

Alkaline Phosphatase↗

PubMed1990

c-fos expression precedes osteogenic differentiation of cartilage cells in vitro.

We have investigated the temporal pattern of expression of c-fos in cartilage cells in mouse mandibular condyles. During in vitro cultivation, the progenitor cells in this organ differentiate to osteoblasts, and hypertrophic chondrocytes start to show features indicative of osteogenic differentiation. Prior to these processes we observed two distinct patterns of c-fos expression. High, transient c-fos expression was found in the entire tissue within 30 min of culture. This type of c-fos expression appeared to result from mechanical forces applied during dissection. The second type of c-fos expression appeared in individual cells in the zone of hypertrophic chondrocytes. A varying number of formerly quiescent chondrocytes expressed high levels of c-fos mRNA after between 30 min and 10 d in culture, with a peak in the number of cells between days 1 and 3. c-fos expression in these cartilage cells was followed by DNA replication and expression of genes typifying osteoblastic differentiation. After 7 d in culture, groups of cells with the typical ultrastructural features of osteoblasts, and surrounded by an osteoid-like matrix, were observed in single chondrocyte-type lacunae, suggesting division of chondrocytes and differentiation to osteoblasts. The data suggest that c-fos may play a crucial role in the perturbation of determined pathways of skeletoblast differentiation and in the regulation of endochondral bone formation.

Animals↗

PubMed1990

Infection of human fibroblasts and osteoblast-like cells with HIV-1.

Primary human skin- and lung-derived fibroblast cell cultures and continuous human osteoblast-like and fibroblast-like cell lines were infected with different strains of HIV-1. Infection was measured at the single-cell level using the immunoperoxidase staining method to detect viral proteins. No cytopathic effects were observed in HIV-1-infected cell cultures. One continuous cell line (LC5), derived from embryonic lung, was readily infectable with HIV-1 and showed continuous production of infectious virus. Infection of LC5 cells could be blocked with anti-CD4 monoclonal antibodies. These findings indicate that fibroblasts of skin and lung, and osteogenic cells may be considered as potential target cells for HIV-1, thereby possibly contributing to the establishment of local HIV reservoirs.

Acquired Immunodeficiency Syndrome↗

PubMed1990

Characterization of fos-induced osteogenic tumours and tumour-derived murine cell lines.

Osteogenic tumours from c-fos (MT-c-fos-LTR)-transgenic mice and from mice infected with the v-fos-bearing FBR murine osteosarcoma virus (FBR MSV) showed close morphological and neoplastic similarities. Fos mRNA expression was elevated in both types of tumours, and expression of several genes characteristic of differentiated bone cells was either lower, enhanced, or not detectable in comparison to that in normal bone. Tumour-derived cell lines showed variable levels of exogenous fos expression; bone-cell-specific genes were similarly expressed in both primary tumours and tumour-derived cell lines. Upon transplantation the tumour cells formed fibrosarcomas, some of which contained areas of focal osseochondrous differentiation. Non-tumorigenic cell lines established from bone tissue of normal and MT-c-fos-LTR transgenic mice showed osteoblastic characteristics, whereas no parathyroid hormone (PTH) response was observed in transgenic tumour cell lines in spite of high alkaline phosphatase activity. These data indicate that deregulated fos expression interferes with terminal osteogenic differentiation in v-fos- and c-fos-induced bone tumours.

Alkaline Phosphatase↗