Search PubMed⌕ Search

Biomedical subjects

S Amselem

Publications and source records attributed to S Amselem.

At least 91 records · Page 5Linked to original sources
PubMed1991

Identification of a new mutation responsible for hepatoerythropoietic porphyria.

A deficiency in the activity of uroporphyrinogen decarboxylase (URO-D), the fifth enzyme of the haem biosynthetic pathway, is found in two hereditary diseases, familial porphyria cutanea tarda (PCT) and hepatoerythropoietic porphyria (HEP). Little is known about the genetic relationship between those two diseases and it has been postulated that HEP is the homozygous form of PCT. A URO-D cDNA was cloned from an HEP patient and the comparison between the mutant and the wild-type sequences showed a single base difference within the coding sequence leading to the replacement of a glutamic acid by a lysine at codon 167 of the mutant protein. This replacement produced a protein which is rapidly degraded in the presence of cell lysate. On the basis of hybridization of synthetic oligomers to amplified genomic DNA, we demonstrated that this patient is homozygous for this single base mutation. In order to look for any relationship between HEP and PCT, we tested six unrelated patients with familial PCT and could not detect the codon 167 mutation in any of them. These results indicate an heterogeneity in the mutations responsible for the PCT and HEP phenotypes.

Amino Acid Sequence↗
PubMed1991

Recurrent nonsense mutations in the growth hormone receptor from patients with Laron dwarfism.

In addition to its classical effects on growth, growth hormone (GH) has been shown to have a number of other actions, all of which are initiated by an interaction with specific high affinity receptors present in a variety of tissues. Purification of a rabbit liver protein via its ability to bind GH has allowed the isolation of a cDNA encoding a putative human growth hormone receptor that belongs to a new class of transmembrane receptors. We have previously shown that this putative growth hormone receptor gene is genetically linked to Laron dwarfism, a rare autosomal recessive syndrome caused by target resistance to GH. Nevertheless, the inability to express the corresponding full-length coding sequence and the lack of a test for growth-promoting function have hampered a direct confirmation of its role in growth. We have now identified three nonsense mutations within this growth hormone receptor gene, lying at positions corresponding to the amino terminal extremity and causing a truncation of the molecule, thereby deleting a large portion of both the GH binding domain and the full transmembrane and intracellular domains. Three independent patients with Laron dwarfism born of consanguineous parents were homozygous for these defects. Two defects were identical and consisted of a CG to TG transition. Not only do these results confirm the growth-promoting activity of this receptor but they also suggest that CpG doublets may represent hot spots for mutations in the growth hormone receptor gene that are responsible for hereditary dwarfism.

Dwarfism↗
PubMed1991

Exclusion of linkage between the collagenase gene and generalized recessive dystrophic epidermolysis bullosa phenotype.

Generalized recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited autosomal disease characterized by dermolytic blister formation. Enhanced collagenase and/or abnormal collagenase have been reported in skin from affected patients, suggesting that collagenase could be responsible for the absence of anchoring fibrils in this disorder. We used a genetic linkage approach to test the hypothesis that this disease is due to a defect in the collagenase gene in nine affected families. Analysis of amplified genomic DNA fragments of the collagenase gene by means of denaturing gradient gel electrophoresis (DGGE) allowed us to detect intragenic polymorphisms, which were subsequently characterized by direct genomic sequencing. Segregation analysis of these polymorphic sites showed exclusion of linkage between the collagenase gene and generalized RDEB phenotype in a family with consanguineous parents and three affected children. However, the possibility of linkage with the collagenase gene in the other eight families tested could not be excluded. The genetic markers described here provide a tool for investigating genetic linkage in other affected families. Overall, our results show that generalized RDEB can be caused by a defect in a gene other than the collagenase gene, and support the hypothesis that the genetic defect lies in abnormal anchoring fibril formation.

Base Sequence↗
PubMed1991

[Molecular genetic of growth hormone resistance syndrome].

The cloning of a putative growth hormone receptor (GH-R) cDNA has opened new approaches for the understanding of the molecular basis of GH insensitivity in humans. This molecule belongs to a new class of transmembrane receptors including prolactin, granulocyte-macrophage colony stimulating factor, erythropoeitin and some interleukin receptors. Although the domains responsible for signal transduction have not yet been identified, the molecular study of a GH-resistance syndrome described by Laron et al. should provide insight into the structure-function relationships of the GH-R and related receptors. This autosomal recessive disorder is characterized by very low serum levels of Insulin-Like Growth Factor I (IGF-I), despite increased secretion of GH with normal activity. Two approaches can be used to test the involvement of the GH-R in this syndrome. The first one, which is indirect, is performed through linkage analysis between GH-R and Laron phenotype; this allowed us to incriminate the GH-R gene in this syndrome. The second approach consists in the identification of molecular defects in the GH-R gene of patients with Laron syndrome; this allowed the detection of a partial gene deletion, two stop codons and one missense mutation. The short stature of the Pygmee population could be related to the Laron syndrome because individuals from this population are also resistant to GH therapy. Therefore, it seems interesting to search for molecular variations of the GH-R gene in this population. Nevertheless, preliminary results indicate that the GH-R gene is not directly involved in this particular short stature condition.(ABSTRACT TRUNCATED AT 250 WORDS)

Black People↗
PubMed1990

Optimization and upscaling of doxorubicin-containing liposomes for clinical use.

This work describes the optimization of a doxorubicin (DXR)-containing liposome formulation and its upscaling for human therapy. Multilamellar vesicles (MLV) composed of egg phosphatidylcholine, egg-derived phosphatidylglycerol, cholesterol, and the drug were prepared in 0.9% sterile, pyrogen-free NaCl by five different hydration methods. The optimal hydration was shown to be the formation of a thin lipid film with high surface area. Alternative hydration methods based on freeze-drying techniques of the lipids in tertiary butanol or based on "alcohol" premixing procedures of the dry DXR-lipid mixture showed smaller DXR loading capacities and lower DXR incorporation per phospholipid. Maximal DXR entrapment was obtained at a molar concentration of phosphatidylglycerol of 30 mol % of total phospholipid. This and previous studies led to a final lipid composition of phosphatidylcholine:phosphatidylglycerol:cholesterol in a molar ratio of 7:3:4. Oligolamellar DXR-liposomes with an average diameter in the range 0.3-0.5 microM were prepared from DXR-MLV by extrusion through polycarbonate membranes using moderate pressures (up to 100 psi). At this size range, maximal entrapment of DXR per phospholipid was obtained. The extrusion process also ensures the sterilization of the final product. Free DXR was removed from liposome-associated DXR (L-DXR) by the use of a cation-exchange resin. The L-DXR formulation was shown to have reasonable stability on storage at 4 degrees C.

Chemistry, Pharmaceutical↗
PubMed1990

A LDL receptor gene homozygous mutation: PCR amplification, direct genomic sequencing, associated haplotype, rapid screening for frequency.

Many mutations in the LDL receptor (LDLR) gene have now been identified mostly as gross gene rearrangements, however they only represent a weak percentage of all deleterious gene mutations causing Familial Hypercholesterolemia (FH). This discrepancy may be related to the difficulties in characterizing point or small defective mutations. In a three-generation family with Familial Hypercholesterolemia, one specific haplotype constructed with 12 intragenic restriction fragment length polymorphisms (RFLP) cosegregated with the disease, while in the consanguineous propositus there was homozygosity for this haplotype. By polymerase chain reaction (PCR) amplification followed by direct sequencing there was unequivocal evidence for a double dose of a unique mutation, (namely a duplication of 4 bases in exon 17), while there was a single dose in heterozygote relatives. We consequently screened a population selected under clinical and geographical criteria for this mutation by PCR and allele specific oligonucleotides (ASO) hybridization. None of the 158 type IIa individuals tested carried the same mutation. Herein, is a rapid combined genetic and molecular approach to characterize and evaluate the frequency of LDL Receptor gene mutations causing Familial Hypercholesterolemia, towards targeted prevention and therapy.

Alleles↗
PubMed1990

Frequent detection of minimal residual disease by use of the polymerase chain reaction in long-term survivors after bone marrow transplantation for chronic myeloid leukemia.

The polymerase chain reaction (PCR) allows the detection of minimal amounts of nucleic sequences and has been successfully used to test for the chronic myeloid leukemia-specific bcr/abl transcripts. We studied blood samples from 17 patients who had undergone allogeneic bone marrow transplantation for CML, using a modified polymerase chain reaction-based assay for the detection of leukemic mRNA. This nested PCR technique was found to be highly sensitive, detecting the chimeric bcr/abl transcript in 16 of 17 patients including several long-term survivors. Cytogenetic techniques failed to detect Ph mitoses. The clinical significance of the persisting bcr/abl transcript for long periods following BMT is poorly understood and remains to be elucidated by further studies.

Bone Marrow Transplantation↗
PubMed1989

Laron dwarfism and mutations of the growth hormone-receptor gene.

Laron dwarfism is associated with resistance to growth hormone (GH). To investigate its genetic basis, we used genetic linkage to test whether the disorder results from a defect in the gene for the human GH receptor. Denaturing gradient gel electrophoresis and sequencing of specific GH-receptor-gene fragments allowed us to characterize specific intragenic DNA markers in 35 control subjects of Mediterranean descent, for use in linkage studies. In two Mediterranean families in which the parents were consanguineous and some of the children had Laron dwarfism, the disease trait and DNA polymorphisms were inherited together. Moreover, an analysis of the GH-receptor-gene RNA transcripts in lymphocytes from one of these families allowed us to identify a thymidine-to-cytosine substitution that generated a serine in place of a phenylalanine at position 96 in the extracellular coding domain of the mature protein. This defect probably affects the receptor adversely and is probably responsible for the lack of plasma GH-binding activity in the patients. This mutation was not found in the GH-receptor genomic sequences of seven unrelated subjects with Laron dwarfism who belonged to different population groups. An analysis of the GH-receptor markers in these patients indicated that different gene frameworks (polymorphic sites within the single gene) were associated with the mutant alleles. We conclude that Laron dwarfism is due to abnormalities in the gene for GH receptor, which may differ from family to family.

Base Sequence↗
PubMed1989

Systemic administration of doxorubicin-containing liposomes in cancer patients: a phase I study.

A clinical study was designed to evaluate the tolerance of cancer patients to liposome-associated doxorubicin (L-DXR). The liposomes used contain phosphatidylglycerol, phosphatidylcholine, cholesterol, and DXR intercalated in the lipid bilayer, and have a mean size in the range of 0.3-0.5 microns. Thirty-two patients, most of them with primary or metastatic liver cancer refractory to conventional therapy, were entered into the study. A total of 69 courses of therapy was administered by intravenous infusion of a suspension of L-DXR (0.5-2.0 mg DXR/ml) in physiologic saline at an approximate rate of 2 ml/min given on a 3-week intermittent schedule. The L-DXR and phospholipid doses were escalated from 20 mg/m2 and 0.3 g/m2 to 120 mg/m2 and 3.2 g/m2 respectively. Treatment was generally well tolerated and acute toxic effects such as nausea and vomiting were mild and infrequent. Chills and fever (greater than 38.0 degrees C) were observed in three patients during infusion of L-DXR and in seven patients 6-12 h after the end of infusion. Median WBC nadir counts were 2700, 2300 and 700/microliters at 85, 100 and 120 mg/m2 respectively. All three patients receiving 120 mg/m2 developed grade 4 leukopenia and fever requiring intravenous antibiotics, and, in two of them, severe stomatitis (grades 3 and 4) was observed. Significant hair loss was apparent in all patients receiving doses higher than 50 mg/m2. The maximal tolerated dose of L-DXR appears to be 120 mg/m2, with leukopenia and stomatitis being the dose-limiting factors. While the subacute toxicity of L-DXR appears to be qualitatively similar to that of free DXR, its tolerance exceeds the recommended dose of free DXR (75 mg/m2) in the standard 3-weekly schedule.

Adult↗
PubMed1989

Mutations in the catalytic domain of human coagulation factor IX: rapid characterization by direct genomic sequencing of DNA fragments displaying an altered melting behavior.

Deficiency in coagulation factor IX, a plasma glycoprotein constituent of the clotting cascade, results in hemophilia B, an inherited recessive X-linked bleeding disorder. Some affected individuals, referred to as antigen positive or CRM+, express an inactive factor IX gene product at normal levels and are expected to have natural mutations altering domains of the molecule that are critical for its correct function. The serine protease catalytic domain of activated factor IX, encoded by exons VII and VIII of the gene, is a possible target for such mutations. We designed a strategy allowing rapid analysis of this region through enzymatic amplification of genomic DNA, analysis of the amplification products by denaturing gradient gel electrophoresis, and direct sequencing of the fragments displaying an altered melting behavior. This procedure permitted us to characterize two previously undescribed mutations. Factor IX Angers is a G-to-A substitution generating an Arg in place of a Gly at amino acid 396 of the mature factor IX protein. Factor IX Bordeaux is an A-to-T substitution introducing a nonsense codon in place of the normal codon for Lys at position 411. Moreover, the already described factor IX Vancouver defect was found in three apparently independent families. These results provide further insight into the molecular heterogeneity of hemophilia B. In addition, we demonstrate the usefulness of this rapid screening procedure, which has broad applications in human genetics and can be used as an alternative to RFLP analysis in carrier detection or prenatal diagnosis studies.

Base Sequence↗
PubMed1989

A 5' splice-region G----C mutation in exon 1 of the human beta-globin gene inhibits pre-mRNA splicing: a mechanism for beta+-thalassemia.

We have characterized a Mediterranean beta-thalassemia allele containing a sequence change at codon 30 that alters both beta-globin pre-mRNA splicing and the structure of the hemoglobin product. Presumably, this G----C transversion at position -1 of intron 1 reduces severely the utilization of the normal 5' splice site since the level of the Arg----Thr mutant hemoglobin (designated hemoglobin Kairouan) found in the erythrocytes of the patient is very low (2% of total hemoglobin). Since no natural mutations of the guanine located at position -1 of the CAG/GTAAGT consensus sequence had been isolated previously, we investigated the role of this nucleotide in the constitution of an active 5' splice site by studying the splicing of the pre-mRNA in cell-free extracts. We demonstrate that correct splicing of the mutant pre-mRNA is 98% inhibited. Our results provide further insights into the mechanisms of pre-mRNA maturation by revealing that the last residue of the exon plays a role at least equivalent to that of the intron residue at position +5.

Adult↗
PubMed1988

DNA haplotype distribution in Algerian beta thalassaemia patients. An extended evaluation by family studies and representative molecular characterization.

An evaluation of beta thalassaemia mutations and the associated chromosomal haplotypes has been made among Algerian thalassaemic patients in this extended series. The major features of our findings are: (i) due to elevated proportion of consanguinity, the frequency of true homozygotes for a defect is high; (ii) Despite this high homozygosity within families, the number of molecular defects resulting in beta thalassaemia are very heterogeneous within this population. This is exemplified not only by the high heterogeneity of haplotypes and associated mutations, but also by the definition of several new haplotypes, among which two of them were found to be associated with novel mutations. Family studies have been performed in parallel to evaluate the degree of feasibility of antenatal diagnosis in this population.

Algeria↗
PubMed1988

Identification of a CpG mutation in the coagulation factor-IX gene by analysis of amplified DNA sequences.

In a family with no previous bleeding history, the sister of a single, severely affected haemophilia B patient requested carrier detection and prenatal diagnosis. In Southern blots, using Taq I digested DNA and a factor-IX cDNA probe, a normal invariant band at 1.6 kb was missing in the haemophiliac suggesting the loss of the Taq I site at the 5' end of exon h. A 162 bp sequence which includes the suspected mutant region was amplified by the polymerase chain reaction in each DNA. Two oligonucleotide probes were synthesized and differed by only one base pair which substituted a T for C in the normal Taq I recognition sequence. The amplified DNA was dot-blotted and hybridized with the labelled probes. The altered sequence hybridized to DNA from the affected individual, his sister and her fetus and not to DNA from the normals. The mutation, involving the haemophiliac, his mother, his sister and her fetus, transforms a CGA codon that encodes for arginine in the catalytic domain of the protein into a UGA stop codon.

Adult↗
PubMed1988

Determination of the spectrum of beta-thalassemia genes in Spain by use of dot-blot analysis of amplified beta-globin DNA.

We have delineated the molecular lesions causing beta-thalassemia in Spain, a country that has witnessed the passage of different Mediterranean populations over the centuries, in order to evaluate the extent of heterogeneity of these mutations and to make possible simplified prenatal diagnosis of the disorder in that country. The use of the polymerase chain-reaction (PCR) technique to preferentially amplify beta-globin DNA sequences that contain the most frequent beta-thalassemia mutations in Mediterraneans enabled us to rapidly analyze 58 beta-thalassemia alleles in a dot-blot format either by hybridization with allele-specific radiolabeled oligonucleotide probes or by direct sequence analysis of the amplification product. The Spanish population carries seven different beta-thalassemia mutations; the nonsense codon 39 is predominant (64%), whereas the IVS1 position 110 mutation, the most common cause of beta-thalassemia in the eastern part of the Mediterranean basin, is underrepresented (8.5%). The IVS1 mutation at position 6 accounts for 15% of the defects and leads to a more severe form of beta+-thalassemia than originally described in most of the patients we studied. In this study, we demonstrate further the usefulness of the dot-blot hybridization of PCR-amplified genomic DNA in both rapid population surveys and prenatal diagnosis of beta-thalassemia.

Base Sequence↗
PubMed1986

Membrane vesicles containing the Sendai virus binding glycoprotein, but not the viral fusion protein, fuse with phosphatidylserine liposomes at low pH.

Membrane vesicles containing the Sendai virus hemagglutinin/neuraminidase (HN) glycoprotein were able to induce carboxyfluorescein (CF) release from loaded phosphatidylserine (PS) but not loaded phosphatidylcholine (PC) liposomes. Similarly, fluorescence dequenching was observed only when HN vesicles, bearing self-quenched N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (N-NBD-PE), were incubated with PS but not PC liposomes. Thus, fusion between Sendai virus HN glycoprotein vesicles and the negatively charged PS liposomes is suggested. Induction of CF release and fluorescence dequenching were not observed when Pronase-treated HN vesicles were incubated with the PS liposomes. On the other hand, the fusogenic activity of the HN vesicles was not inhibited by treatment with dithiothreitol (DTT) or phenylmethanesulfonyl fluoride (PMSF), both of which are known to inhibit the Sendai virus fusogenic activity. Fusion was highly dependent on the pH of the medium, being maximal after an incubation of 60-90 s at pH 4.0. Electron microscopy studies showed that incubation at pH 4.0 of the HN vesicles with PS liposomes, both of which are of an average diameter of 150 nm, resulted in the formation of large unilamellar vesicles, the average diameter of which reached 450 nm. The relevance of these observations to the mechanism of liposome-membrane and virus-membrane fusion is discussed.

Animals↗
PubMed1986

Fusion of Sendai virus with negatively charged liposomes as studied by pyrene-labelled phospholipid liposomes.

Sendai virus particles fuse with negatively charged liposomes but not with vesicles made of zwitterionic phospholipids. The liposome-virus fusion process was studied by dilution of the concentration-dependent excimer-forming fluorophore 2-pyrenyldodecanoylphosphatidylcholine contained in the liposomes by the viral lipids. The data were analyzed in the framework of a mass action kinetic model. This provided analytical solutions for the final levels of probe dilution and numerical solutions for the kinetics of the overall fusion process, in terms of rate constants for the liposome-virus adhesion, deadhesion and fusion. This analysis led to the following conclusions: At neutral pH and 37 degrees C, only 15% of the virus particles can fuse with the phospholipid vesicles, although all the virions may aggregate with the liposomes. The rate constants for aggregation, fusion and deadhesion are of the orders of magnitude of 10(7) M-1 X s-1, 10(-3) s-1 and 10(-2), s-1, respectively. The fraction of active virus increases with temperature. At acidic pH, both the fraction of 'fusable' virus and the rate of fusion increase markedly. The optimal pH for fusion is 3-4, where most of the virus particles are active. At higher pH values, an increasing fraction of the virus particles become inactive, probably due to ionization of viral glycoproteins, whereas at pH values below 3.0 the fusion is markedly reduced, most likely due to protonation of the negatively charged vesicles. While only 15% of the virions fuse with the liposomes at pH 7.4 and 37 degrees C, all the liposomes lose their content (Amselem, S., Loyter, A. Lichtenberg, D. and Barenholz, Y. (1985) Biochim. Biophys. Acta 820, 1-10). We therefore propose that release of entrapped solutes is due to liposome-virus aggregation, and not to fusion. Both trypsinization and heat inactivation of the virus particles inhibit not only the fusion process but also the release of carboxyfluorescein. This demonstrates the obligatory role of viral membrane proteins in liposome-virus aggregation. Reconstituted vesicles made of the viral lipid and the hemagglutinin/neuraminidase (HN) glycoprotein fuse with negatively charged liposomes similar to the intact virions. This suggests that the fusion of virions with negatively charged vesicles, unlike the fusion of the virus with biological membranes, requires only the HN and not the fusion glycoprotein.

Calcium↗
PubMed1985

The interaction of Sendai virus with negatively charged liposomes: virus-induced lysis of carboxyfluorescein-loaded small unilamellar vesicles.

The interaction of Sendai virus with small, unilamellar vesicles, lacking virus receptors and loaded with self-quenched 6-carboxyfluorescein, was studied. Sendai virions induced release of carboxyfluorescein from vesicles composed of negative charged phospholipids, despite the fact that they did not contain virus receptors. Preliminary experiments indicate that the carboxyfluorescein release is accompanied by mixing of the virus and liposome lipids and their entrapped contents, suggesting liposome-virus fusion. No release of carboxyfluorescein was observed with vesicles containing only phosphatidylcholine. The rate of virus-induced carboxyfluorescein release was temperature dependent; the lytic activity of the virus was greatly enhanced above 25 degrees C. This effect was not due to a thermal phase transition of the lipids in either the lipid vesicles or the virions. Virus-induced carboxyfluorescein release was inhibited by the presence of calcium ions in the medium and of cholesterol in the lipid vesicles. It increased with increasing concentrations of either the lipid vesicles or the virions. pretreatment of virions with increasing concentrations of three different proteolytic enzymes (trypsin, chymotrypsin and proteinase) inhibited the virus' ability to cause release of carboxyfluorescein from negatively charged liposomes. Inhibition of the viral lytic activity was also observed after virions were incubated above 56 degrees C.

Calcium↗