Biomedical subjects

R Benarous

Publications and source records attributed to R Benarous.

At least 55 records · Page 3Linked to original sources

PubMed1994

Monoclonal antibody-mediated inhibition of RNA binding and annealing activities of HIV type 1 nucleocapsid protein.

Retroviral nucleocapsid (NC) proteins are highly basic, with one or two zinc fingers, and are required for virion formation, genomic RNA dimerization and packaging, and replication primer tRNA annealing to the viral RNA. We report here the first characterization of monoclonal antibodies directed against a retroviral nucleocapsid protein and their use to study the structure-function relationships of the nucleocapsid protein NCp7 of HIV-1. Four anti-NCp7 monoclonal antibodies (MAbs) have been generated by using NCp7 of HIV-1. The epitope targets of these MAbs were mapped using ELISA and BIAcore techniques. Whereas three of them are specific for epitopes located in the N and C termini of NCp7, the fourth one appears to be conformation specific. Interestingly, only two of these MAbs, the conformation-specific one and the MAb recognizing an N-terminal epitope are able to inhibit the RNA-binding and annealing activities of NCp7 as well as strong-stop DNA synthesis in vitro. The binding of the two other MAbs onto NCp7 either has no effect or enhances the NCp7-RNA interactions. These MAbs also display a differential recognition of the Gag polyprotein precursor, which makes them useful tools for studying NC protein maturation in the course of virion morphogenesis.

Amino Acid Sequence↗

PubMed1994

Decreased expression of the two D2 dopamine receptor isoforms in bromocriptine-resistant prolactinomas.

Bromocriptine or other dopamine agonists are usually effective for the treatment of prolactin-secreting adenomas. Five to 18% of prolactinomas, however, do not respond to such therapy. We have shown previously that such resistance to bromocriptine correlates with reduced binding to the D2 receptor subtype of dopamine, the major PRL inhibiting factor. In the present work, we demonstrated that reduced binding actually corresponds to decreased expression of the gene coding for the D2 receptor in the pituitary from bromocriptine-resistant patients, as shown by 4-fold lower levels of the corresponding mRNAs compared to those coding for actin. The existence of two D2 receptor isoforms, D2S and D2L generated by alternative splicing, has been described in several tissues, including the pituitary. Both are negatively coupled to adenylyl cyclase and inhibit prolactin secretion, but, in addition, the shortest one (D2S) is more efficiently coupled to phospholipase C. Consequently, we also investigated whether expression of a particular D2 receptor isoform was preferentially affected in resistant adenomas. The proportion of messengers corresponding to the short receptor isoform (D2S) was lower in resistant compared to responsive adenomas: D2S/D2L = 0.74 +/- 0.08 and 1.00 +/- 0.07, respectively. In parallel, much lower levels of D2 receptor mRNAs were found in growth hormone-secreting adenomas, with a D2S/D2L ratio comparable to those of both normal human pituitary and bromocriptine-sensitive prolactinomas (1.05 +/- 0.11). Thus, resistance to bromocriptine therapy seems to involve defects in D2 dopamine receptor expression and possibly in posttranscriptional splicing.

Adenoma↗

PubMed1994

P56lck A lymphocyte specific protein tyrosine kinase: activation, regulation and signal transduction.

p56lck, a src family protein tyrosine kinase interacts with several T cell receptors, like: CD4, CD8, CD2 and the beta-chain of the IL2, thereby receptors devoid of kinase activity may transduce signals via tyr phosphorylation. Tyr 192 and ser 194, located in the SH2 domain of p56lck is phosphorylated upon CD3 triggering, which can change interactions of tyr-P proteins with this SH2 domain. Upon activation through the CD2 or the CD45 receptors the kinase activity of p56lck is temporarily increased. By immunofluorescent and confocal microscopy we observed that a significant proportion of p56lck and CD2 receptors are localized in endosomal vesicles after stimulation. By Western blot we showed a parallel recruitment of the PTK p70-ZAP in this vesicles. The role of p56lck away from the plasma membrane localized in vesicles is under study.

CD2 Antigens↗

PubMed1994

Pattern of expression of five alternative transcripts of the lck gene in different hematopoietic malignancies: correlation of the level of lck messenger RNA I B with the immature phenotype of the malignancy.

The lck gene encodes a tyrosine protein kinase of the src family which is highly expressed in T-lymphocytes. Two widely separated promoters govern expression of the lck gene. We report in this study that alternative splicing between cryptic donor and acceptor sites in the 5' untranslated region of the transcripts initiated at the type I promoter give rise to three different type I lck mRNAs, I A, I B, and I C. Altogether with the types II A and II B reported previously, the lck transcription is thus characterized by five alternative transcripts. We further used the complementary DNA-polymerase chain reaction assay to describe the pattern of expression of these five lck transcripts in different hematopoietic cell lines and in blood or bone marrow samples from healthy donors and leukemic patients. We report that the transcript II A is by far the major transcript present both in human samples and in T-cell lines. The low lck expression in B-cell lines is characterized by the quite exclusive presence of the transcript I B. We show that hematopoietic diseases characterized by the presence of immature cells [acute myeloid leukemia (AML-0 and AML-1) and T- and B-cell acute lymphoid leukemia] exhibit a marked increase of the transcript I B. No significant difference from the normal pattern is observed in AML according to the differentiation stage (AML-2 to AML-5). A normal pattern of lck expression is restored in AML-0 and AML-1 patients at complete remission.

Adolescent↗

PubMed1993

Characterization and regulation of the angiotensin II type-1 receptor (binding and mRNA) in human adrenal fasciculata-reticularis cells.

The classical concept of human adrenal physiology indicates that only glomerulosa cells are the target of A-II. Herein, we demonstrated that cultured human adrenal fasciculata-reticularis cells were also responsive to this hormone. Indeed, these cells contained high affinity (Kd = 0.9-1.1 nM) and low capacity (8,000-13,000 sites/cell) A-II receptors, and more than 95% of them were of the type-1. These AT1 receptors are functional since A-II was able to increase cortisol production after 48 h of treatment. These effects were inhibited by losartan, an AT1 antagonist, but not by CGP42112A, an AT2 antagonist. The expression of the type-1 A-II receptor mRNA was detected in the whole adrenal in both adult and fetus, and in cultured human adrenal fasciculata-reticularis cells. In these cells A-II negatively regulated AT1 receptor mRNA, and this effect was also mediated through the AT1 receptor subtype.

Adult↗

PubMed1993

Detection of Toxoplasma gondii in venous blood from AIDS patients by polymerase chain reaction.

Detection of Toxoplasma gondii in blood by means of the polymerase chain reaction (PCR) may facilitate the diagnosis and follow-up of cerebral toxoplasmosis in patients with AIDS. We evaluated this approach with seven patients with tissue culture-proven parasitemia, 14 patients with presumptive cerebral toxoplasmosis, and 17 healthy human immunodeficiency virus-positive controls. Each sample of blood was assayed on three different occasions by a PCR assay based on detection of the gene encoding the P30 surface protein. A positive PCR diagnosis required positivity in at least two of the three PCR tests. None of the controls had a positive PCR diagnosis, but six of the seven patients with parasitemia did. Cerebral toxoplasmosis was confirmed in 13 of the 14 patients with a presumptive diagnosis; diagnosis by PCR was positive before treatment for 9 of these 13 patients, whereas tissue culture was positive for only 1 patient. During treatment, blood samples were taken from 14 patients at regular intervals until day 12. PCR diagnosis became negative on ethidium-stained gels, but persistent signals were observed after hybridization, in some cases, for up to 12 days after initiation of therapy. PCR on venous blood could thus be a sensitive and noninvasive method for the diagnosis of cerebral and disseminated toxoplasmosis in AIDS patients and could be a potential tool for monitoring the effects of treatment.

AIDS-Related Opportunistic Infections↗

PubMed1993

Recombinant Csk expressed in Escherichia coli is autophosphorylated on tyrosine residue(s).

The C-terminal src kinase (Csk) is responsible for the phosphorylation of the carboxy-terminal tyrosine of several tyrosine kinases of the Src family. This phosphorylation site has a negative regulatory function. Csk is unique among the members of the protein tyrosine kinase family because it lacks the conserved tyrosine autophosphorylation site and has been thought to be devoid of autophosphorylation activity. Using the glutathione S-transferase (GST) bacterial expression system, we have produced large amounts of a chimeric rat Csk protein. We have determined that the GST-Csk fusion protein isolated from bacteria is autophosphorylated on tyrosine residue(s). GST-Csk and purified Csk are capable of undergoing autophosphorylation on tyrosine residue(s) in vitro. The GST-Csk fusion protein also phosphorylates exogenous substrates, including the heteropolymer poly-Glu/Tyr and enolase. This is the first indication that Csk is autophosphorylated on tyrosine residue(s) both in vivo in bacteria expressing Csk cDNA and in vitro. These findings suggest that the autophosphorylation of Csk might play a role in the regulation of its kinase activity as well as its binding to other cellular proteins.

Antibodies, Monoclonal↗

PubMed1993

Selective increase of alternatively spliced Lck transcripts from the proximal promotor in hematopoietic malignancies.

Screening by Northern blot for lck expression in 51 patients with diverse hematologic diseases has shown, for four of them, a 3 to 15-fold increase in the level of lck mRNA when compared with expression in healthy donors. These patients suffered from diverse malignancies: one Burkitt lymphoma, one T-cell lymphoma and two non-Hodgkin B-cell lymphoma. Specific analysis of the different lck transcripts in these patients by polymerase chain reaction and their relative quantitation demonstrate a significant increase of only the type IB and the type IC lck transcripts arising from the proximal promotor. Our study shows: (a) a high lck expression may occur in diverse hematologic diseases, (b) whatever the type of malignancy, this high expression results in a specific increase of the spliced transcripts arising only from the proximal promotor, and (c) in these four patients without any rearrangement or amplification, the high lck expression probably results from the specific activation of the proximal promotor.

Blotting, Northern↗

PubMed1992

Spleen protein tyrosine kinases TPK-IIB and CSK display different immunoreactivity and opposite specificities toward c-src-derived peptides.

Polyclonal antibodies have been raised against two synthetic peptides reproducing the 48-64 and 353-369 sequences of CSK, a protein tyrosine kinase implicated in the down-regulation of src-related protein kinases. Both antibodies specifically recognize recombinant CSK and a CSK-related 49 kDa protein tyrosine kinase present in spleen but they do not cross-react with purified TPK-IIB, a spleen protein tyrosine kinase sharing with CSK catalytic activity toward src kinases and incapability to autophosphorylate. CSK and TPK-IIB once resolved from each other by heparin-Sepharose affinity chromatography, display opposite specificities toward synthetic peptides reproducing the sequences around the main phosphoacceptor residues of pp60c-src, namely Tyr-416 and Tyr-527. These data support the view that TPK-IIB and CSK may exert opposite effects on the activity of src-related protein tyrosine kinases.

Amino Acid Sequence↗

PubMed1992

Alternative splicing in the human lck gene leads to the deletion of exon 1' and results in a new type II lck transcript.

In this study we describe a new lck mRNA transcribed from the type II promoter, resulting from alternative splicing, in the deletion of exon 1' which comprises the AUG initiation codon. In this type IIB lck transcript, as a consequence of exon 1' deletion, a new AUG codon in exon 1, located 35 nt upstream from the regular initiation codon in exon 1', and normally out of frame, is now in frame with exon 2 and the following lck coding sequence. In this type IIB lck transcript, 10 residues encoded by exon 1 from the new AUG codon replace the first 35 residues encoded by exon 1'. Strikingly, in this new N-terminal amino acid sequence, a glycine residue, needed for myristylation and anchorage in the plasma membrane, is present at position 2. This alternative splicing has been observed in T-cell lines, normal mature T cells and in peripheral blood lymphocytes from different leukemic patients. Different ratios of regular type IIA lck mRNA (with exon 1') to spliced type IIB lck mRNA (without exon 1') have been observed depending on the patient.

Alternative Splicing↗

PubMed1992

Comparative value of polymerase chain reaction and conventional biological tests for the prenatal diagnosis of congenital toxoplasmosis.

When toxoplasmosis is acquired during pregnancy, there is a risk of severe congenital defect in the foetus. Maternal treatment with spiramycin limits the transplacental passage of the parasite to the foetus but does not prevent infection in all cases. Prenatal diagnosis should be based on specific and fast methods to prescribe the more potent combination of sulfadiazine and pyrimethamine. This study evaluates PCR in the prenatal diagnosis of toxoplasmosis; PCR was based on the detection of the gene coding for the P30 surface protein. Amniotic fluid from 44 women with suspected foetal infection was tested by PCR and results were compared to those of conventional diagnostic tests on foetal blood and amniotic fluid. PCR was positive in 7 out of 10 samples from proven congenital toxoplasmosis cases. Sensitivity of PCR was similar to cell culture and mouse inoculation of amniotic fluid but was superior to tests carried out on foetal blood (specific IgM, eosinophil and platelet counts, gamma glutamyl transferase, mouse inoculation). In two cases, PCR was positive with no detected infection of the foetus. In this study, the combination of fast detection methods, ie cell culture and PCR of amniotic fluid, eosinophil and platelet counts, GGT activity and specific IgM, enabled us to confirm 10/10 cases of congenital toxoplasmosis in less than a week. PCR therefore appears to be an additional test which improves early prenatal diagnosis of toxoplasmosis.

Amniotic Fluid↗

PubMed1991

Identification and expression of the cDNA of KIN17, a zinc-finger gene located on mouse chromosome 2, encoding a new DNA-binding protein.

We report the cloning of KIN17 cDNA, 1414 bp long with an ORF of 391 residues showing a zinc finger and nuclear localization signals. By recloning the cDNA into an appropriate vector, we produced kin17 protein in E. coli, purified it partially and shown that kin17 protein binds to double-stranded DNA. The KIN17 gene was localized by cytogenetic mapping in mouse chromosome 2, band A. Genomic sequences homologous to KIN17 cDNA were detected also in rat and human DNAs. KIN17 mRNA is highly expressed in rodent transformed AtT-20 neuroendocrine cells whereas it can be detected only in the total RNA of mouse embryos and various normal adult tissues by reverse transcription and PCR amplification. The mouse nuclear kin17 protein was identified by a local small structural similarity with E.coli recA protein. Kin17 and recA have only 39 amino acid residues in a region that might be involved in DNA-binding.

Amino Acid Sequence↗

PubMed1990

Partial apolipoprotein E-beta-galactosidase fusion protein expressed in Escherichia coli retains binding activity to the LDL(B/E) receptor.

A partial rat apo E-beta-galactosidase fusion protein was produced in Escherichia coli Y1089 infected with recombinant lambda GT11 obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum. Partial cDNA overlapped the apo E mRNA sequence coding for apo E binding domain towards the LDL(B/E) receptor up to codon for Arg-139. Fusion protein specifically bound to human fibroblasts. The high-affinity component exhibited a Kd of 5 x 10(-8) M and 4.1 x 10(5) sites per cell. Fusion protein binding to fibroblasts was mediated by their apo E moiety and not by beta-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) beta-galactosidase did not compete with fusion protein binding; and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in LDL(B/E) receptor, bound fusion protein 10-times lower than control fibroblasts. It was demonstrated that partial fusion protein retained the functional activity of the native apo E. However, compared to full-length native or engineered apo E, fusion protein was able to bind fibroblasts without being complexed with phospholipids. Fusion proteins might be a useful tool for studying the functional efficiency of the LDL(B/E) receptor and for mapping residues and domains involved in the binding process.

Apolipoproteins E↗

PubMed1990

Tissue-plasminogen activator RNA detected in megakaryocytes by in situ hybridization and biotinylated probe.

Tissue-plasminogen activator (t-PA) has been identified in human megakaryocytes isolated from human rib fragments or from culture cells. The presence of t-PA antigen in the endoplasmic reticulum of mature cells was demonstrated by ultrastructural immunochemistry. These results suggest that t-PA can be synthesized by megakaryocytes as well as by vascular endothelial cells. In order to confirm this possibility, in situ hybridization using antisense RNA biotinylated probe was used in megakaryocyte culture at different steps of maturation. The presence of t-PA-RNA was clearly demonstrated at an early step of differentiation. Biotin staining was enhanced in mature cells and this was correlated with the detection of t-PA antigen by immunocytochemistry.

Cells, Cultured↗

PubMed1990

Identification of two human ferritin H genes on the short arm of chromosome 6.

We have found by analyses of human-hamster hybrid cells that two human ferritin H genes lie near the locus of the iron storage disease idiopathic hemochromatosis on chromosome 6p. One of these genes was isolated and shown to be a processed pseudogene. Comparison of its sequence with those of other ferritin H pseudogenes indicates that they may be derived from a functional H gene other than that on chromosome 11.

Animals↗

PubMed1989

Structure of the human lck gene: differences in genomic organisation within src-related genes affect only N-terminal exons.

Although cDNA sequences coding for several Rous sarcoma virus Src-related protein tyrosine kinases (PTKs) have been reported for several years, knowledge of the structure and organisation of genes of the src family is still limited. In this work, a detailed structure and organisation of the human lck gene is reported. A 17-kb genomic clone encoding human p56 Lck, a lymphocyte-specific PTK of the Src-related subfamily, has been isolated. The human lck gene is organized in 13 exons, one more than in the human cellular (c)-src gene. The twelve coding exons are located in this clone, whereas the putative 5'-noncoding exon is probably located very far upstream from the second exon. Splicing sites for exons 4 to 12, which encode both conserved phospholipase-C-like and catalytic domains of the Src-like PTKs, arise exactly at the same position for the human lck, human c-src and c-fgr genes. The only differences concern the splice sites of exons 1' and 2, which encode the unique N-terminal domain of human Lck. These results give further evidence that the different PTKs of the Src-like family have probably evolved through the mechanism of exon shuffling.

Amino Acid Sequence↗

PubMed1989

Apolipoprotein-E-gene expression in rat liver during development in relation to insulin and glucagon.

An apolipoprotein-E (apo-E) cDNA probe, cloned by immunoscreening of a lambda GT11 rat liver cDNA library, was used to further characterize the expression of the apo-E gene in rat liver during development, in relation to plasma insulin and glucagon levels. The apo-E mRNA level was low in fetus liver, then abruptly increased at birth and rose further during the suckling period. It returned to the level at birth in 10-week-old adults. These variations were paralleled with dramatic changes in plasma glucagon, which rose at birth and remained high during suckling. At the same time, the insulin/glucagon molar ratio fell. Administration of N6,O2-dibutyryl cAMP to 5-day-old rats resulted in a significant induction of liver apo-E mRNA. Moreover, liver apo-E mRNA rose in 10-h-fasted suckling rats as compared to controls, while plasma glucagon increased and the insulin/glucagon ratio decreased. Conversely, glucose feeding of suckling rats did not induce any increase in liver apo-E mRNA, the insulin/glucagon ratio was 10-fold higher than in fasted animals. Our results are consistent with liver apo-E gene expression being under the control of plasma glucagon and of the glucagon/insulin balance.

Animals↗

PubMed1989

Multiple mRNAs encode peripherin, a neuronal intermediate filament protein.

Three cDNA clones of 1.6 (3u), 1.2 (5g) and 0.6 (5b) kbp, specific for peripherin, a neuronal intermediate filament protein (IFP), have been isolated from a murine neuroblastoma cell lambda gt11 library by immunoscreening using peripherin antiserum. Antibodies eluted from the fusion proteins produced by clones 3u and 5g recognize the peripherin spots on immunoblots. Where they overlap the three cDNAs have identical sequences. cDNA 5g exhibits the closest homology to type III IFP cDNAs. cDNA 3u is identical to the corresponding region of cDNA 5g, except for the insertion of a 96 bp fragment at a position corresponding to the junction of exons 4 and 5 in type III IFP cDNAs. cDNA 5b is also identical to the corresponding region of cDNA 5g, except for the deletion of a 62 bp fragment at the junction of exons 8 and 9 in type III IFP cDNAs. S1 mapping experiments performed with probes covering the 3' end of the two unexpected regions show that three distinct mRNAs correspond to the three cDNAs. Moreover, three peripherin products, two minor 61 and 56 kd products in addition to the major 58 kd peripherin, are observed when poly(A)+ RNA is in vitro translated, the 61 kd peripherin being translated from the 3u-selected RNA. The three RNAs originate from alternative splicing of a unique peripherin gene, thus generating polymorphism of peripherin.

Amino Acid Sequence↗