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M Antoniou

Publications and source records attributed to M Antoniou.

At least 37 records · Page 2Linked to original sources
PubMed1995

Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene.

Retrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and sickle cell anemia), however, has been hampered by the inability to generate recombinant viruses able to efficiently and faithfully transmit the necessary sequences for appropriate gene expression. We have addressed this problem by carefully examining the interactions between retroviral and beta-globin gene sequences which affect vector transmission, stability, and expression. First, we examined the transmission properties of a large number of different recombinant proviral genomes which vary both in the precise nature of vector, beta-globin structural gene, and locus control region (LCR) core sequences incorporated and in the placement and orientation of those sequences. Through this analysis, we identified one specific vector, termed M beta 6L, which carries both the human beta-globin gene and core elements HS2, HS3, and HS4 from the LCR and faithfully transmits recombinant proviral sequences to cells with titers greater than 10(6) per ml. Populations of murine erythroleukemia (MEL) cells transduced by this virus expressed levels of human beta-globin transcript which, on a per gene copy basis, were 78% of the levels detected in an MEL-derived cell line, Hu11, which carries human chromosome 11, the site of the beta-globin locus. Analysis of individual transduced MEL cell clones, however, indicated that, while expression was detected in every clone tested (n = 17), the levels of human beta-globin treatment varied between 4% and 146% of the levels in Hu11. This clonal variation in expression levels suggests that small beta-globin LCR sequences may not provide for as strict chromosomal position-independent expression of beta-globin as previously suspected, at least in the context of retrovirus-mediated gene transfer.

Animals↗
PubMed1995

The seroprevalence of ten zoonoses in two villages of Crete, Greece.

The seroprevalence and incidence of 10 zoonoses due to Rickettsia typhi, R. conorii, Coxiella burnetii, Burcella sp., Borrelia sp., Toxoplasma sp., Leishmania sp., Entamoeba histolytica, Echinococcus granulosus and Fasciola hepatica were studied in an animal husbandry and a farming village in Crete, Greece. The serum conversion incidence of each infectious agent was determined by testing 2 blood samples, collected in 1985 and in 1987. The surveillance was conducted using detailed transparent maps of the 2 villages studied, on which epidemiological data were interrelated to the results obtained from the serological tests. Thus the importance and spread of each infection were visualized. C. burnetii, Toxoplasma sp., R. conorii, and E. granulosus, were the most common infectious agents encountered during this study.

Adult↗
PubMed1995

Delivery of DNA into mammalian cells by receptor-mediated endocytosis and gene therapy.

The correction of genetically based disorders by the introduction of a therapeutic genetic construct into the appropriate cell type ("gene therapy"), has become a distinct possibility in recent years. In order for gene therapy to be a practical alternative to more conventional pharmaceutical approaches to treatment, it must be administrable in vivo. This demands that a system be developed that can specifically target the DNA to the desired cell type once introduced into the patient. Among the procedures that are currently being pursued, the delivery of DNA to cells by receptor mediated endocytosis (RME), comes closest to fulfilling this crucial requirement. The natural physiological process of RME can be exploited to deliver genetic material to cells. An antibody or ligand to a cell surface receptor that is known to undergo endocytosis, is complexed with DNA through a covalently linked polycationic adjunct (e.g., polylysine, protamines). Such complexes retain their binding specificity to the cell surface and are taken up into the cell where they enter the endosomal compartment via normal endocytotic processes. In addition, steps must be taken to avoid degradation of the DNA within the endosome-lysosome. Cells can be treated with the lysosomatropic agent chloroquine during the transfection procedure. Alternatively, the components of viruses that enter cells by endocysis and possess an endosomal "break out" capacity can be used. Replication defective adenovirus coupled to the ligand-DNA complex gives transfection efficiencies of virtually 100% on tissue culture cells in vitro. Synthetic peptides that mimic the membrane fusing region of influenza virus hemagglutinin, have also been successfully used as part of the ligand-DNA complex to bring about endosomal escape. Preliminary studies have demonstrated the potential of this method to specifically target DNA to the cell type of choice in vivo. Delivery of genes by receptor-mediated endocytosis offers the greatest hope that gene therapy can be an inexpensive, easily applicable, widespread technology.

Animals↗
PubMed1995

Regulated high-level human beta-globin gene expression in erythroid cells following recombinant adeno-associated virus-mediated gene transfer.

Gene therapy approaches for beta-thalassemia and sickle cell anemia focus on the transfer of a human beta-globin gene into the patient's hematopoietic stem cells (HSC). Expression of the transferred sequences should be erythroid specific and match the expression of the endogenous alpha-globin genes in adult erythropoiesis. Here we explore the potential of recombinant adeno-associated virus (AAV) vectors for human beta-globin gene transfer. We have constructed a recombinant AAV-vector containing a human beta-globin gene together with the DNasel hypersensitive sites 4, 3 and 2 of the human beta-globin locus control region. The vector replicates to high titers and can efficiently transduce hematopoietic and non-hematopoietic cells. In transduced and G418 selected murine erythroleukemia (MEL) cell clones, human beta-globin gene expression was regulated and reached levels comparable to endogenous murine beta maj. These data show that AAV-vectors are promising tools in gene therapy approaches for the haemoglobinopathies.

Animals↗
PubMed1994

Transcriptional activation by hypersensitive site three of the human beta-globin locus control region in murine erythroleukemia cells.

In this paper we describe a complete deletional analysis of hypersensitive site three (HS3) of the human beta-globin Locus Control Region (LCR). The previously defined core fragment consists of 6 footprinted regions, with multiple binding sites for the erythroid-specific factor GATA-1 and G-rich motifs that can interact with ubiquitous factors such as Sp1 and TEF-2. We show in this paper that the 5' half of this fragment is the most important for activity in murine erythroleukemia (MEL) cells. A fragment containing footprints 1-4 can stimulate transcription of a linked human beta-globin gene to levels of about 40% of that obtained with footprints 1-6. Constructs containing either footprints 1-3 or 3-6 cannot be distinguished from the beta-globin gene alone. We further show that binding sites for the erythroid-specific factor NF-E2 can co-operatively interact with parts of the HS3 core fragment, and that HS3 requires elements upstream from -103 in the human beta-globin promoter for full activity. The importance of these results is discussed in the context of the regulation of the genes in the human beta-globin cluster.

Animals↗
PubMed1994

Synthesis and biological activity of NK1 substance P selective agonists by modifying the methionyl residue.

A series of analogues of substance P, where the Met11 residue was replaced by Glu(OCH2CH3), Glu(OBzl) and Hse(CH3), were synthesized in order to investigate the effect on agonist activity of modifications at the C-terminal residue. The biological activities in the guinea-pig ileum assay (NK1 receptor) and rat [text says rabbit] pulmonary artery (NK2 receptor) indicate that replacement of the SCH3 group of Met11 of substance P by the COOBzl or OCH3 groups favour interaction with the NK1 receptor and increase selectivity towards this receptor subtype.

Amino Acid Sequence↗
PubMed1994

Synthesis and biological activity of NK1 tachykinin antagonists not containing D-residues.

A series of analogues of the previously reported NK1 tachykinin antagonist [Orn6, Glu(OBzl)11]-SP6-11-OBzl has been synthesized and tested in order to investigate the effects on antagonistic activity of modifications at the C-terminal residue. The biological activity in the guinea-pig ileum assay (NK1 receptor) indicates that the two aromatic rings introduced in the C-terminal part of the peptide are both essential for the expression of antagonistic activity.

Amino Acid Sequence↗
PubMed1994

Analogues of the C-terminal fragments of neurokinins with modifications at their C-terminal methionyl residue. Structure-activity studies.

Analogues of SP4-11 have been synthesized in which the methionyl residue is replaced successively by the Glu(OCH2CH3), Glu(OBzl), Hse(CH3) and Glu(CONHCH3) residues, and analogues of NKA4-10 and NKB4-10 have been prepared in which the methionyl residue is replaced by the Hse(Bzl) and Hse(CH3) residues, respectively. The SP4-11 analogues were tested in three in vitro preparations representative of NK-1, NK-2 and NK-3 receptor types. Substitution of the SCH3 group of the Met11 side chain by the groups COOCH2CH3 and COOBzl has little affect on the agonist activity in NK-1 preparations, while in NK-2 the corresponding analogues are more potent than the parent octapeptide; that substituted with COOBzl being 8.2 times more potent than SP4-11. In NK-3 preparations all analogues are weak agonists. The selectivity of all the analogues is reduced compared with the corresponding hexapeptide analogues. The SP4-11 analogues, along with those of NKA4-10 and NKB4-10, were tested for their binding ability in the three receptor subtypes above. The SP4-11 analogues show reduced affinity for NK-1 receptors, while the NKA4-10 and NKB4-10 analogues have almost the same affinities as NKA and NKB for NK-2 and NK-3 receptors, respectively. The effect of the lipophilicity of the Met11 side chain, especially when a phenyl group is present in the side chain, at the NK-2 receptor is discussed.

Amino Acid Sequence↗
PubMed1993

The regulation of human globin gene switching.

This paper describes the mechanism of regulation of the human beta-globin on the basis of a number of natural mutations and experiments in transgenic mice. From these data we conclude that this multigene locus is regulated at a number of different levels involving specific interactions between the Locus Control Region (LCR) and the individual genes. Most important is the action of stage specific transcription factors acting on sequences immediately flanking the genes. In addition, specificity is obtained through specific interaction of the genes with the LCR and through competition of the genes for interaction with the LCR.

Animals↗
PubMed1993

Studies on Echinococcus granulosus using the scanning electron microscope. I. Preparations of the parasite for infection of the final host.

The development of the protoscoleces of Echinococcus granulosus was studied after chemical stimulation, which mimics that offered by the final host. This stimulation resulted in some morphological changes in the parasite and to its evagination. These changes prepare the parasite to infect the intestine of its final host. The external morphology of the protoscolex body and of the germinal layer of the cyst and brood capsule were studied using the scanning electron microscope (SEM). Findings were related to other studies using the transmission electron microscope (TEM). The morphology of the flame cell is described and four cases of teratogenesis are reported.

Animals↗
PubMed1993

Studies on Echinococcus granulosus using the scanning electron microscope. II. The hooks.

The morphology of the hooks of Echinococcus granulosus at the protoscolex level was studied by means of scanning electron microscopy. Hooks were uncovered in situ and also freed from the rostellum so as to study them in detail. There were two types of hooks: those of the upper row, which were larger and had a projecting, rounded stout guard, and those of the lower row, which were smaller and had a flattened guard. These and other differences in the morphology of the two types of hooks were studied and related to the differences in their function.

Animals↗
PubMed1993

Nuclear organization of splicing snRNPs during differentiation of murine erythroleukemia cells in vitro.

Murine erythroleukemia (MEL) cells are erythroid progenitors that can be induced to undergo terminal erythroid differentiation in culture. We have used MEL cells here as a model system to study the nuclear organization of splicing snRNPs during the physiological changes in gene expression which accompany differentiation. In uninduced MEL cells, snRNPs are widely distributed throughout the nucleoplasm and show an elevated concentration in coiled bodies. Within the first two days after induction of terminal erythroid differentiation, the pattern of gene expression changes, erythroid-specific transcription is activated and transcription of many other genes is repressed. During this early stage splicing snRNPs remain widely distributed through the nucleoplasm and continue to associate with coiled bodies. At later stages of differentiation (four to six days), when total transcription levels have greatly decreased, splicing snRNPs are redistributed. By six days postinduction snRNPs were concentrated in large clusters of interchromatin granules and no longer associated with coiled bodies. At the end-point of erythroid differentiation, just before enucleation, we observe a dramatic segregation of splicing snRNPs from the condensed chromatin. Analysis by EM shows that the snRNPs are packaged into a membrane-associated structure at the nuclear periphery which we term the "SCIM" domain (i.e., SnRNP Clusters Inside a Membrane).

Animals↗
PubMed1993

Each hypersensitive site of the human beta-globin locus control region confers a different developmental pattern of expression on the globin genes.

We have tested the effect of the individual DNase I hypersensitive site (HS) regions of the globin locus control region (LCR) on the developmental expression pattern of the human gamma and beta genes in transgenic mice. The results show that HS3 is the most active site during the embryonic period. It is also the only site capable of high level expression of the gamma genes during fetal hematopoiesis, in a population of cells that are capable of expressing both the gamma and beta genes. Region HS4 shows the highest activity during the adult stage and expresses the gamma genes only at low levels during the embryonic period. HS2 drives equivalent levels of gamma or beta transgene expression throughout development. HS1 has a similar pattern to HS2, although the activity of HS1 is very low. From these results we conclude that the HS regions have distinct developmental specificities and suggest that in the complete LCR they interact with each other to form a larger complex which, in turn, interacts with the globin genes.

Animals↗
PubMed1993

Regulated expression of K+ channel genes in electrically silent mammalian cells by linkage to beta-globin gene-activation elements.

Fundamental studies of the mechanism of human beta-like globin gene-expression have identified DNA sequences (locus control regions or LCRs) which directly influence the specific activation of beta-globin genes in erythroid cells. Here we report the first applications of LCR-mediated gene-activation to stable electrophysiological expression of several homomultimeric ion channel proteins. We describe expression driven from a native K+ channel gene promoter region, contiguous to an intronless coding sequence, within a single excised human genomic DNA fragment. In addition, cDNAs encoding both inactivating and non-inactivating mammalian K+ currents have been expressed by insertion between the promoter and second intron of the human beta-globin gene. Expression levels are characteristically independent of integration position and are proportional to LCR-gene copy number, a parameter specific for each clonal cell line. K+ channel expression is inducible by erythroid differentiation and has been demonstrated by electrophysiological recordings of cells taken directly from culture.

Amino Acid Sequence↗
PubMed1992

LCR/MEL: a versatile system for high-level expression of heterologous proteins in erythroid cells.

We have used the human globin locus control region (LCR) to assemble an expression system capable of high-level, integration position-independent expression of heterologous genes and cDNAs in murine erythroleukaemia (MEL) cells. The cDNAs are inserted between the human beta-globin promoter and the second intron of the human beta-globin gene, and this expression cassette is then placed downstream of the LCR and transfected into MEL cells. The cDNAs are expressed at levels similar to those of the murine beta-globin in the induced MEL cells. Heterologous genomic sequences can also be expressed at similar levels when linked to to the LCR and beta-globin promoter. In addition we demonstrate that, after induction of differentiation, MEL cells are capable of secreting heterologous proteins over a prolonged time period, making this system suitable for use in continuous production systems such as hollow fibre bioreactors. The utility of the LCR/MEL cell system is demonstrated by the expression of growth hormone at high levels (greater than 100 mg/l) 7 days after induction. Since the expression levels seen do not depend upon gene amplification and are independent of the integration position of the expression cassette, it is possible to obtain clones with stable high-level expression within 3-4 weeks after transfection.

Animals↗
PubMed1992

Synthesis and biological activity of analogues of the C-terminal hexapeptide of substance P with modifications at glutaminyl and methioninyl residues. Structure-activity studies.

Analogues of [Orn6]-SP6-11 have been synthesized in which the methionyl residue is replaced by glutamine gamma-carboxamide substituted derivatives. These analogues where tested in three in vitro preparations representative of NK-1, NK-2 and NK-3 receptor types. Substitution of the SCH3 group of the Met11 side chain by CONHCH3, CON(CH3)2, CONHPh and CONCH3Ph groups results in analogues which are full agonists in NK-1 and NK-2 preparations with the exception of the Glu[N(CH3)2]11 and the Glu(NHCH3)11 analogues, which are partial agonists at NK-1 and NK-2 receptors respectively. The Glu(NHCH3)11 analogue shows selectivity for the NK-1 receptor type and is equipotent to the Glu(NCH3Ph)11 analogue in the same receptor type. The latter analogue is 2.84 times more potent than the parent hexapeptide in the NK-2 preparation. The Glu(NHPh)11 analogue is a full agonist in the NK-3 preparation and equipotent to the parent hexapeptide, in contrast to the other analogues in which Met has been replaced by glutamine gamma-carboxamide substituted residues. It is concluded that for NK-1 receptor type the lipophilic character of Met11 side chain is not a determining factor for activity but it is an important factor for activity in the NK-2 receptor type and has a stronger effect when a phenyl group is present, thus leading to analogues which are full agonists and more potent than the parent hexapeptide.

Amino Acid Sequence↗
PubMed1990

Definition of the minimal requirements within the human beta-globin gene and the dominant control region for high level expression.

The human beta-globin dominant control region (DCR) was previously identified as a region from the 5' end of the human beta-globin locus which directs high level, site of integration-independent, copy number-dependent expression on a linked human beta-globin gene in transgenic mice and stably transfected mouse erythroleukaemia (MEL) cells. We have now analysed the elements comprising the DCR by systematic deletion mutagenesis in stable MEL transfectants. We have identified two independent elements within the DNase I hypersensitive sites 2 and 3, containing fragments which direct strong transcriptional inducibility of a beta-globin gene. Whilst the remaining two hypersensitive sites do not direct significant transcriptional induction, our data suggest that all four sites may be necessary for the fully regulated expression conferred by the DCR. We have also tested a number of beta-globin minigene constructs under the control of the DCR to assess if any of the local sequences from the gene may be removed without loss of expression. We find that the 3' enhancer may be removed without affecting expression, but there is an absolute requirement for the presence of the second intron, not related to the enhancer present in that intron.

Animals↗