Biomedical subjects

V Erfle

Publications and source records attributed to V Erfle.

At least 73 records · Page 4Linked to original sources

PubMed1994

Splicing variability in HIV type 1 revealed by quantitative RNA polymerase chain reaction.

A quantitative RNA-polymerase chain reaction (PCR) method able to detect the majority of mRNAs produced by human immunodeficiency virus type 1 (HIV-1) was developed and used to study expression of different HIV-1 clones in human cells. Amplified mRNAs were compared to known cDNA standards. This comparison permitted the optimization of PCR conditions and eliminated the generation of artifactual PCR bands. The use of RNA and cDNA standards demonstrated that the RNA amplification is linear within the tested range and suggested that it can be used to quantitate individual mRNAs. The results demonstrate the overall conservation of splicing in different HIV-1 clones. Although, in general, splicing was conserved, extensive qualitative and quantitative variability was observed in different HIV-1 clones. This variability is likely one determinant of the biological characteristics of the different HIV-1 clones, and demonstrates a great plasticity of the HIV-1 genome. The described RNA-PCR methodology was used for the study of HIV-1 expression in unstimulated peripheral blood mononuclear cells (PBMCs) of infected individuals. In general, the same mRNAs were identified in HIV-infected cultured cell lines and in unstimulated PBMCs. Analysis of a variant band found after amplification of PBMC RNA from an HIV-infected individual revealed a new splice site for the generation of Rev/Nef-encoding mRNAs. The availability of a sensitive, rapid, and essentially quantitative method to examine the major HIV-1 mRNAs will facilitate the detailed analysis of HIV-1 expression in human cells.

Base Sequence↗

PubMed1994

[Murine leukemia virus Akv promotes bone tumor development in fos-transgenic mice].

Newborn hMT-fos-LTR transgenic C3H mice and their non-transgenic siblings were infected with Akv, derived from the ecotropic provirus of the AKR mouse. Bone sarcomas in non-infected transgenics were observed in 20% (3/15) of females at 448 +/- 25 days and in 8% (1/12) of males at 523 days. Akv-infected transgenics developed bone tumors with higher frequency and at younger age: Females in 69% (20/28) at 268 +/- 122 days, males in 83% (24/29) at 279 +/- 109 days. In the majority of the bone tumors of Akv-infected transgenics (70% in females, 59% in males) cellular atypia was lacking and the histological pattern resembled human parosteal osteosarcoma. Only 50% (12/24) of bone tumors in Akv-infected transgenics revealed newly integrated virus sequences by Southern analysis. PCR analysis detected Akv sequences in DNAs of all tumors. Obviously, the insertion of Akv in a few cells induced the considerably accelerated bone tumor growth.

Animals↗

PubMed1993

Endogenous superantigen expression controlled by a novel promoter in the MMTV long terminal repeat.

Endogenous superantigens are encoded by the open reading frame contained within the mouse mammary tumour virus long terminal repeat (MMTV LTR). Superantigen expression results in T-cell proliferation and, during early ontogeny, T-cell deletion. Here we identify a novel promoter located upstream of the previously described MMTV promoter. Transcripts from this promoter initiate within the U3 region of the MMTV LTR and splice to the acceptor for endogenous superantigen coding region. The novel U3 promoter is active in B lymphocytes, which are cognate antigen-presenting cells for endogenous superantigen, and is able to direct expression of superantigen in the absence of the previously described MMTV promoter.

Animals↗

PubMed1993

Large scale production and purification of human retrovirus-like particles related to the mouse mammary tumor virus.

Human retrovirus-like particles related to mouse mammary tumor virus (MMTV) are secreted in a steroid-dependent manner by the breast cancer cell line T47D. We report the successful large scale production and purification of these particles from culture supernatants of T47D cells and describe the experimental conditions established for this purpose. Thus, mg amounts of particles were produced by large scale culturing of T47D cells in an autoharvesting roller bottle system and purified by differential centrifugation and continuous flow ultracentrifugation on density gradients with a 50% recovery and a 350-fold enrichment.

Animals↗

PubMed1993

Genomic distribution and transcription of solitary HERV-K LTRs.

The human genome contains a family of endogenous retroviruses, HERV-K, with sequence homology to the B-type mouse mammary tumor virus. We have now identified a single HERV-K LTR within the C-type-related human retroviral element S71. The HERV-K LTR is located in the antisense direction between the S71 gag and the pol gene, replacing the 5' half of S71 pol. A number of HERV-K LTR-related cDNA clones were detected by screening various human cDNA libraries with an S71 HERV-K LTR probe, indicating abundant transcription of HERV-K-related LTRs in human tissues. Sequence analysis of four cDNA clones revealed LTR sequences with a nucleotide identity of 70 to 90% with HERV-K10 LTR. Some HERV-K-related LTR sequences contain potential short open reading frames. The analyzed cDNA clones do not harbor any retroviral sequences other than those related to HERV-K LTRs. However, most of the solitary LTRs were found to be coexpressed with cellular sequences. Transcription of these LTRs is probably directed by external cellular promoters. We show that HERV-KLTR-like sequences entered the primate genome about 33-40 million years ago. We estimate the human genome to contain about 25,000 copies of HERV-K-related LTRs, which are distributed over most human chromosomes in an irregular manner.

Animals↗

PubMed1993

Productive expression state confers resistance of human immunodeficiency virus (HIV)-2-infected lymphoma cells against superinfection by HIV-1.

In the past, positive as well as negative results pertaining to HIV-1/HIV2 interference have been obtained. Therefore, in the present study attention was paid to the viral expression state of preinfected cells at the time of exposure to secondary virus. A clonal HIV-2 infected HUT-78 cell line was derived by endpoint dilution and subsequently inoculated with cell-free HIV-1. Superinfection with HIV-1 was ruled out by Western blot and PCR analysis. The chronically HIV-2 infected cells used for these studies showed a highly productive expression state, as evidenced by immunoperoxidase staining (IPS), Western blot profile and levels of reverse transcriptase (RT) activity. We discuss several mechanisms of interference in productively infected cells, which may confer resistance to superinfection with secondary virus.

Blotting, Southern↗

PubMed1993

Bryodin, a single-chain ribosome-inactivating protein, selectively inhibits the growth of HIV-1-infected cells and reduces HIV-1 production.

Bryodin, a single-chain ribosome-inactivating protein (RIP) isolated from Bryonia cretica ssp dioica (cucurbitaceae), was found to selectively inhibit the growth of persistently HIV-1-infected T lymphoma cells (KE37/1) and human lung fibroblast when used in concentrations from 2-20 micrograms/ml. Uninfected KE37/1 cells remained unaffected at the same doses of bryodin. In addition, bryodin reduced HIV production in the surviving infected cells. Two isoforms of bryodin were purified by dye ligand chromatography. Both isoforms exerted the growth-inhibiting influence and reduced HIV production. Trichosanthin, another member of the RIP family, had similar inhibitory effects on the growth of HIV-1 infected cells and on HIV-1 production. Bryodin and trichosanthin were effective in about the same dose range. No selective effects for HIV-infected cells were observed with the RIPs gelonin and ricin.

Cells, Cultured↗

PubMed1993

Reduction of HIV-1 antigen production by phosphatidylcholine containing formulations via growth inhibition of HIV-1-infected cells.

Phosphatidylcholine (PC) and licensed formulations containing PC were tested for their influence on the proliferation and viability of cells permanently infected with HIV-1 (human immunodeficiency virus type 1). PC alone, as well as pharmaceutical formulations containing PC, selectively inhibited the growth of productively infected lymphoid cells. The strongest growth inhibition was observed with formulations containing PC, glycerol and triglyceride together. The growth inhibition was dose-dependent for HIV-1-infected cells. Additionally, PC-containing formulations dramatically reduced antigen production from peripheral blood mononuclear cells (PBMCs) infected in vitro with HIV-1. In vivo experiments with Rauscher-MuLV-infected mice showed that PC administered either intraperitoneally or orally was able to inhibit Rauscher-virus-induced splenomegaly. PC-containing formulations are currently used in man for supportive therapy at doses, which in vitro induced 50% growth inhibition of HIV-1-infected cells in vitro. Such doses have been used in man without side effects for many years. Thus, PC-containing formulations may be valuable for the treatment of HIV-1-infected individuals.

Animals↗

PubMed1993

Sub-retroviral particles as gene transfer vectors that by-pass retrovirus-receptor interaction restrictions.

Retroviral infection initiates with the binding of the virus envelope protein to cell membrane receptors. However, it is unclear whether this interaction is necessary for subsequent events required to establish infection. To address this problem subretroviral particles containing a retroviral vector genome carrying a beta-galactosidase (beta-gal) gene have been microinjected into cells. Here we show that such cells become infected as judged by expression of beta-gal activity, suggesting that virus-receptor interaction is not required for infection. Such subretroviral particles lacking an envelope may be useful both for safer gene transfer procedures and gene transfer to cells that do not express the cognate virus receptor.

3T3 Cells↗

PubMed1992

Influence of amphipathic peptides on the HIV-1 production in persistently infected T lymphoma cells.

The effects of several amphipathic peptides on HIV-1 production in persistently infected cells are described. Melittin, a 26 amino acid alpha-helical amphipathic peptide, reduces HIV-1 production dose-dependently, whereas other amphipathic peptides do not. Six melittin derivatives which retain the alpha-helical portion have similar effects as melittin. The reduction of viral infectivity is not due to an effect of melittin on the virus particles but to an intracellular action of the peptide, which is readily taken up into cells, as shown by quantitative ELISA. Western blots of cells from melittin-treated cultures suggest that the processing of the gag/pol precursor is impaired.

Amino Acid Sequence↗

PubMed1992

Exposure of HIV-infected cells to phospholipid leads to membrane alterations and selective growth retardation.

The effect of exogenous phosphatidylcholine on structure and function of plasma membranes from HIV-1-producing cells and from their non-infected counterparts was determined. The membrane protein composition was not affected by phospholipid treatment. Membrane fluidity and Ca(2+)-permeability were increased in virus-producing cells and in control cells after lipid treatment. The triacylglycerol content of the plasma membranes was increased in virus-producing cells after lipid treatment, whereas the content of phospholipid and cholesterol was not changed. The increased triacylglycerol content was in accordance with a relatively higher rate of [14C]oleic acid incorporation into triacylglycerols of the virus-producing cells after lipid treatment as shown by metabolic labeling. The results suggest that a latent cytopathic effect of HIV-infection becomes manifest if the cells are exposed to exogenous phospholipid and this may open a way to preferentially eliminate HIV-producing cells.

Calcium↗

PubMed1992

Elevated p53 RNA expression correlates with incomplete osteogenic differentiation of radiation-induced murine osteosarcomas.

An important role for the p53 gene in osteogenic sarcomas has been imputed by identification of somatically acquired gene alterations in human osteosarcomas and by the development of osteosarcomas in p53 transgenic mice. To study the involvement of p53 in radiation-induced osteosarcomagenesis, we have investigated gene alterations and expression of p53 in radiation-induced murine osteosarcomas and tumor-derived cell lines. Eighteen of 31 tumors and 8 of 9 cells lines showed alterations in the p53 gene region, or elevated levels of p53 RNA. Expression of the osteoblast marker gene bone gla protein was substantially reduced in tumors which simultaneously showed high steady-state levels of p53 RNA. Our data indicate that p53, in addition to its function in regulating DNA synthesis, may be involved in the control of osteogenic differentiation in osteosarcomagenesis.

Animals↗

PubMed1992

Growth inhibition of HIV-1-infected cells and membrane alterations induced by phosphatidylcholine.

Cultured T-lymphoma cell lines H9 and KE37-1 permanently infected with human immunodeficiency virus (HIV-1, strain HTLV-III B) were exposed to phosphatidylcholine (PC) and a PC-containing formulation "Essentiale" (PC-E). PC and PC-E, but not triglyceride, were found to inhibit growth of virus-infected cells. Additionally, the membrane lipid composition of infected and uninfected H9 cells was investigated upon exposure to PC. The HIV-1-infected cells showed a 25% increase in membrane triglyceride content and a 15% increase in membrane phospholipid saturated fatty acids. In the presence of PC, there is a further increase in triglyceride content up to 180% compared with uninfected control cells, suggesting a possible cause for the selective growth inhibition of HIV-1-infected cells by PC. The PC-E dose range effective in vitro for inhibition of HIV-1-infected cell growth falls within the range that can be reached in vivo. Formulations containing PC are well tolerated by humans and might be applicable at an early stage of HIV-1 infection to reduce the number of virus-producing cells.

Cell Division↗

PubMed1992

Cellular localization of Nef expressed in persistently HIV-1-infected low-producer astrocytes.

OBJECTIVES: The characterization and localization of HIV-1 Nef highly expressed in permanently infected astrocytes (TH4-7-5) as a model for latent infection of human brain cells. DESIGN: Immunochemical methods are an appropriate tool to investigate expression and localization of cellular proteins. METHODS: Nef expression was analysed by Western blot and immunoperoxidase staining using a panel of monoclonal and polyclonal antibodies. Cellular localization studies were performed by indirect immunofluorescence and subcellular fractionation of TH4-7-5 cells. Myristoylation of Nef was investigated by immunoprecipitation of [3H]myristic acid-labelled cell extract. TH4-7-5 nef gene was cloned and amplified by polymerase chain reaction and the nef nucleotide sequence analysed. RESULTS: Reactivities of various Nef-specific antibodies with Nef antigen in TH4-7-5 cells were demonstrated by Western blot analysis. Immunofluorescence revealed cytoplasmic perinuclear staining of Nef with most antibodies. However, one monoclonal antibody against amino acids 168-175 of Nef showed intense homogeneous nuclear staining in TH4-7-5 cells. Reactivity of this Nef antibody was blocked with recombinant Nef derived from TH4-7-5 cells. After subcellular fractionation, Nef was detected in nuclear, membrane and cytosolic fractions of TH4-7-5 cells. No myristoylated Nef antigen was detectable, perhaps because of a serine residue at position 2 of the TH4-7-5 nef gene instead of the glycine residue required for myristoylation. CONCLUSIONS: Chronically HIV-1-infected astrocytoma cells with restricted virus production express different antigenic forms of Nef, which can be distinguished by their subcellular localization. Variant subcellular targeting of Nef suggests the existence of multiple activities of Nef within HIV-infected cells.

Amino Acid Sequence↗

PubMed1992

Retrovirus-like particles from the human T47D cell line are related to mouse mammary tumour virus and are of human endogenous origin.

Retrovirus-like particles were secreted in a steroid-dependent manner by the human mammary carcinoma cell line T47D. The particles exhibited typical retroviral properties such as their electron microscopic appearance (95 nm in diameter) and occasional budding, sedimentation at 1.14 g/ml, reverse transcriptase activity and genomic RNA. The T47D particles were related to mouse mammary tumour virus (MMTV) as shown by their ultrastructural appearance (B type-like eccentric dense cores and budding), Mg2+ dependence of the reverse transcriptase activity; immunological reactivity with MMTV-directed antibodies (revealing proteins of 63K, 52K, 26K and 18K), and hybridization of particle RNA with MMTV DNA under stringent conditions. Purified particles were able to incorporate deoxynucleoside triphosphates in the absence of an exogenous primer and template, thus indicating the existence of a complete and biochemically functional reverse transcription apparatus (reverse transcriptase, RNA and primer) and the ability to direct endogenous cDNA synthesis. Labelled particle cDNA hybridized strongly to human genomic DNA but not to mouse and cat DNA, thus indicating the human origin of the T47D particles. Furthermore all human DNAs, hybridized with the labelled particle cDNA, showed a uniform hybridization pattern of restriction fragments, indicating the endogenous origin and distribution of the proviral particle DNA in the human genome.

Adenocarcinoma↗

PubMed1992

Molecular cloning of osteoma-inducing replication-competent murine leukemia viruses from the RFB osteoma virus stock.

We report the molecular cloning of two replication-competent osteoma-inducing murine leukemia viruses from the RFB osteoma virus stock (M. P. Finkel, C. A. Reilly, Jr., B. O. Biskis, and I. L. Greco, p. 353-366, in C. H. G. Price and F. G. M. Ross, ed., Bone--Certain Aspects of Neoplasia, 1973). Like the original RFB osteoma virus stock, viruses derived from the molecular RFB clones induced multiple osteomas in mice of the CBA/Ca strain. The cloned RFB viruses were indistinguishable by restriction enzyme analysis and by nucleotide sequence analysis of their long-terminal-repeat regions and showed close relatedness to the Akv murine leukemia virus.

Animals↗

PubMed1992

Expression and biological significance of human endogenous retroviral sequences.

The human genome contains a variety of elements resembling mammalian retroviruses. Most of these sequences have been found to be related to primate and murine C-type viruses (BaEV, SSAV/GaLV, MuLV), murine B-type viruses and A-type particles (MMTV, IAP), or human T-cell lymphotropic viruses (HTLV). Altogether, human endogenous retroviruses and retroviral elements are estimated to comprise at least 0.1 to 0.6% of the human genome. Like other transposable elements they may contribute in shaping the eukaryotic genome by intracellular transposition events or by generating hot spots of recombination. Human retroviral sequences have been shown to be transcriptionally active, especially in human placenta and embryonic tissue and in human tumor cell lines. Some elements that are coexpressed with cellular sequences are supposed to play a role in regulation of gene expression. Furthermore, expression of human endogenous retroviral sequences may have a protective function against superinfection by related exogenous retroviruses. On the other hand, endogenous retroviruses and retroviral elements represent a cellular reservoir of possibly pathogenic retroviral genes. They may be involved in chromosomal aberrations by acting as sites for recombination events between different chromosomes. Furthermore, they can act as insertion mutagens and activate or inactivate cellular genes. Retroviral gene products themselves may also be pathogenic as has been shown for the immunosuppressive effects of p15E envelope proteins. Therefore, the role of human endogenous retroviruses and retroviral sequences in biological processes is currently a subject of great interest.

Animals↗

PubMed1992

Metastatic growth of v-fos-induced osteosarcoma, following in vitro cultivation.

A tumour cell line was established from a non-metastatic osteosarcoma which had developed in the right femur after infection of a newborn mouse with FBR osteosarcoma virus (FBR MSV). After 20 cell culture passages and injection of the cells into newborn syngeneic mice, the animals developed fibrosarcomas on the site of injection. Metastatic lung tumours were detected in 50% of the mice. Metastatic tumour growth was preferentially observed in the walls of large blood vessels and in subpleural zones. Large areas of the metastatic tumours resembled mature connective tissue. This model of in vitro progression from a primary non-metastasizing osteosarcoma to a neoplasm with particular metastatic potency, though with a growth pattern similar to fibromatosis may serve as a useful system to study the different steps of metastatic progression, distinct tropism of invasive cells and fibrous maturation of metastatic tumours.

Animals↗