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N Blin

Publications and source records attributed to N Blin.

At least 73 records · Page 4Linked to original sources
PubMed1995

Identification of a receptor/G-protein contact site critical for signaling specificity and G-protein activation.

Each G protein-coupled receptor recognizes only a distinct subset of the many structurally closely related G proteins expressed within a cell. How this selectively is achieved at a molecular level is not well understood, particularly since no specific point-to-point contact sites between a receptor and its cognate G protein(s) have been identified. In this study, we demonstrate that a 4-aa epitope on the m2 muscarinic acetylcholine receptor, a prototypical Gi/o-coupled receptor, can specifically recognize the C-terminal 5 aa of alpha subunits of the Gi/o protein family. The m2 receptor residues involved in this interaction are predicted to be located on one side of an alpha-helical receptor region present at the junction between the third intracellular loop and the sixth transmembrane domain. Coexpression studies with hybrid m2/m3 muscarinic receptors and mutant G-protein alpha q subunits showed that the receptor/G-protein contact site identified in this study is essential for coupling specificity and G-protein activation.

Adenylyl Cyclases↗
PubMed1995

Coexpression pattern of c-myc associated genes in a small cell lung cancer cell line with high steady state c-myc transcription.

The c-Myc protein is involved in cellular transformation and mitogenesis, but also works as a potent inducer of differentiation and programmed cell death. Max as an obligate heterodimeric partner for Myc mediates its functions as a specific transcriptional activator and a transforming protein. Mad and Mxi1 proteins both heterodimerize with Max and compete with each other for limiting amounts of Max. Transcriptional activation by Myc can be suppressed by increasing the amount of Mad or Mxi1. This report shows the expression pattern of these Myc related factors at the mRNA level in a small cell lung cancer (SCLC) cell line (GLC4) which is characterized by c-myc amplification and strong constitutive c-myc overexpression. We found these genes transcriptionally active but uninfluenced from high c-myc transcription. Max was constantly transcribed at a relatively low level during cell cycle progression. Mad and mxi1 mRNA was at a surprisingly high level in proliferating cells. Mad was further upregulated and mxi1 was downregulated to basal levels during serum starvation of the cells. We further analyzed the activity of c-fos, c-jun, c-myb and nm23 which are described to be involved in c-myc transcriptional activation, c-jun and c-fos were not constitutively activated and can be excluded as regulators. High steady state c-myc in contrast influences the serum stimulated transient activation mechanism of these two genes. We identified high copy number nm23 mRNA whose role as a putative c-myc transcriptional activator is under investigation. Our results indicate that constitutive overexpression of c-myc does not require the activity of the nuclear oncogenes tested and that the m-RNA expression pattern of functionally related proteins is not influenced.

Base Sequence↗
PubMed1995

Mapping of single amino acid residues required for selective activation of Gq/11 by the m3 muscarinic acetylcholine receptor.

Each G protein-coupled receptor can interact only with a limited number of the many structurally similar G proteins expressed within a cell. This study was undertaken to identify single amino acids required for selectively coupling the m3 muscarinic acetylcholine receptor to G proteins of the Gq/11 family. To this goal, distinct intracellular segments/amino acids of the m3 receptor were systematically substituted into the structurally closely related m2 muscarinic receptor, which couples to Gi/o proteins, not Gq/11 proteins. The resultant mutant receptors were expressed in COS-7 cells and studied for their ability to induce agonist-dependent stimulation of phosphatidylinositol hydrolysis, a response known to be mediated by G proteins of the Gq/11 class. Using this approach, we were able to identify four amino acids in the second intracellular loop and four amino acids at the C terminus of the third intracellular loop of the m3 muscarinic receptor that are essential for efficient Gq/11 activation. We could demonstrate that these amino acids, together with a short segment at the N terminus of the third intracellular loop, fully account for the G protein coupling preference of the m3 muscarinic receptor. Taken together, our data strongly suggest that only a limited number of amino acids, located on different intracellular regions, are required to determine the functional profile of a given G protein-coupled receptor.

Amino Acid Sequence↗
PubMed1995

Assignment of Alu-repetitive sequences to large restriction fragments from human chromosomes 6 and 22.

We have employed a pulsed field gel electrophoresis and Alu hybridization approach for identification of large restriction fragments on chromosome 6 and 22. This technique allows large portions of selected human chromosomes to be visualized as discrete hybridization signals. Somatic cell hybrid DNA which contains chromosome 6 or chromosome 22 was restricted with either Notl or Mlul. The restriction fragments were separated by pulsed field gel electrophoresis (PFGE) and hybridized against an Alu repetitive sequence (Blur 8). The hybridization signals result in a fingerprint-like pattern which is unique for each chromosome and each restriction enzyme. In addition, a continuous pattern of restriction fragments was demonstrated by gradually increasing puls times. This approach will also be suitable to analyze aberrant human chromosomes retained in somatic cell hybrids and can be used to analyze flow sorted human chromosomes. To this end, our method provides a valuable alternative to standard cytogenetic analysis.

Chromosome Mapping↗
PubMed1995

Muscarinic acetylcholine receptors: structural basis of ligand binding and G protein coupling.

Muscarinic acetylcholine receptors (m1-m5) were studied by a combined molecular genetic/pharmacologic approach to elucidate the molecular characteristics of the ligand binding site and of the receptor domains involved in G protein coupling. Site-directed mutagenesis studies of the rat m3 muscarinic receptor suggest that the acetylcholine binding domain is formed by a series of hydrophilic amino acids located in the "upper" half of transmembrane domains (TM) III, V, VI, and VII. Moreover, we showed that mutational modification of a TM VI Asn residue (Asn507 in the rat m3 receptor sequence) which is characteristic for the muscarinic receptor family has little effect on high-affinity acetylcholine binding and receptor activation, but results in dramatic reductions in binding affinities for certain subclasses of muscarinic antagonists. The N-terminal portion of the third intracellular loop (i3) of muscarinic and other G protein-coupled receptors has been shown to play a central role in determining the G protein coupling profile of a given receptor subtype. Insertion mutagenesis studies with the rat m3 muscarinic receptor suggest that this region forms an amphiphilic alpha-helix and that the hydrophobic side of this helix represents an important G protein recognition surface. Further mutational analysis of this receptor segment showed that Tyr254 located at the N-terminus of the i3 loop of the m3 muscarinic receptor plays a key role in muscarinic receptor-induced Gq activation. The studies described here, complemented by biochemical and biophysical approaches, should eventually lead to a detailed structural model of the ligand-receptor-G protein complex.

Amino Acid Sequence↗
PubMed1995

Lack of isodisomy for chromosome 22 in disomic meningiomas.

Loss of one copy of chromosome 22 is the most prevalent chromosomal change in meningioma, indicative of a tumor suppressor on chromosome 22. Meningioma retaining both copies of chromosome 22 could be possibly be explained by isodisomy for a meningioma suppressor gene. To investigate whether the chromosomal situation in meningioma is consistent with this hypothesis, we studied 53 cases, using polymorphic probes localized on chromosome 22. loss of one copy of chromosome 22 was found in 14 cases when polymorphic DNA markers were used. Thirty-nine meningiomas studied by karyotyping and molecular probes retained both copies of chromosome 22. The majority of cases (30/39, or 77%) displayed heterozygous banding patterns, indicating the presence of heterologous copies of chromosome 22. Hence, our data provide no evidence for duplication of one parental homolog as a general mechanism in diploid meningioma.

Chromosome Aberrations↗
PubMed1995

Predictive quantitative structure-activity relationships (QSAR) analysis of beta 3-adrenergic ligands.

A novel quantitative structure-activity relationships strategy was used to analyze seventeen beta-adrenergic ligands for which we had previously evaluated pharmacological properties in Chinese hamster ovary cells transfected with the human beta 1-, beta 2- or beta 3-adrenergic gene (Blin et al., 1993, Mol. Pharmacol., 44: 1094-1104). These ligands were classified into pharmacological activity categories in order to determine the extent to which molecular structural features may be involved in the selectivity of the interaction with the beta 3-AR, or to define molecular features and properties characteristic of a beta 3-AR high affinity ligand or of a potent beta 3-adrenergic agonist. Topological and physico-chemical molecular descriptors were obtained using a novel software combining calculations with multivariate statistical methods, such as principal component analysis and discriminant analysis. This study showed that beta 1/beta 2-antagonists beta 3-agonists could be differentiate from beta 1/beta 2/beta 3-agonists on the basis of their topological molecular descriptors weighted by partial atomic charge and lipophilicity logP values. Bulky lipophilic groups at the end of the alkylamine chain and an ethoxy function, extending the flexible portion of the molecule and modifying the electron density distribution, were requirements for selective agonism at the beta 3-site. Charge and logP weighted 2D-autocorrelation vectors were properties able to discriminate between classes of agonists in terms of their affinity, potency or intrinsic activity, thus emphasizing the part these molecular descriptors play in determining beta 3-adrenergic ligands. These results, in association with the powerful activity-prediction model evaluated in the test, provide a framework to rationalize the synthesis of new beta 3-AR specific compounds.

Adrenergic Agents↗
PubMed1994

Expression pattern of breast-cancer-associated protein pS2/BCEI in colorectal tumors.

Recently, several carcinomas of the gastrointestinal tract were tested for pS2/BCEI activity, a gene isolated from breast-cancer cells and coding for a small secreted peptide. In the latter tumors, its activity is under estrogen control; surprisingly, it was also found expressed in carcinomas of the stomach, biliary tract and pancreas. We have now investigated the expression of this gene in 64 colorectal carcinomas, 31 adenomas and 13 polyps in comparison with their matrix tissues by applying molecular (RNA analysis) and immunohistochemical (pS2 antibody) techniques. Positive pS2 immunostaining (ranging from focal to strong immunoreaction) was noted in 89% of human colon cancers, while 11% remained negative. Furthermore, all 40 transitional mucosae were strongly positive, whereas normal mucosa was negative. Of hyperplastic polyps, 68.2% displayed a significant immunoreaction, and 80.6% of adenomas were focally positive. Finally, 6 out of 16 cases showed significant pS2 transcription in Northern blot analysis. These data clearly indicate that the breast-cancer-associated pS2 protein also plays an as yet undetermined role in the tumorigenesis of human colorectal carcinomas.

Breast Neoplasms↗
PubMed1994

Telomere length variation in normal and malignant human tissues.

Tissue and tumor specific length variation of telomere (TTAGGG)n repeats was studied in DNAs from various normal and malignant tissues. DNA was isolated from bone marrow and blood cells, malignant tissues, and established tumor cell lines. Nonisotopic Southern hybridization revealed a reduction of telomere repeat arrays in 14 of the 35 tumors analyzed. However, other cases (60%) showed no reduction, or even an increase, in telomeric length. Our finding of elongated telomere stretches in several tumors of different origin compared with normal tissue is in contrast to previous reports describing a general shortening of terminal repeat length in colorectal cancer and neuroblastoma. We tentatively conclude that there is no general tendency to telomere reduction in malignant tissues.

Adolescent↗
PubMed1994

Debrisoquine hydroxylase gene polymorphism in meningioma.

Cytochrome P450 CYP2D6 polymorphism is an autosomal recessive trait associated with impaired debrisoquine metabolism in 5-10% of caucasian populations. This polymorphism has been associated with susceptibility to Parkinson's disease, bladder cancer, various forms of leukemia and possibly melanoma. In many other cancer forms, the data remained contradictory due to the technical limitations for identifying affected individuals (poor metabolizers). A recently developed polymerase chain reaction-based assay allows convenient screening of approximately 80% of known mutations. We have tested brain tumors correlated with chromosome 22 deviations for genetic polymorphism in the cytochrome P450 CYP2D6 locus localized on chromosome 22q13. Thirty-one meningioma samples were analyzed and the observed frequency of heterozygotes and homozygotes for the G to A mutation did not deviate significantly from the distribution in a normal population. These data are comparable to previous observations in for example breast and colon cancer and indicate that the CYP2D6 locus on chromosome 22q13 is not involved in the pathogenesis of meningiomas.

Alleles↗
PubMed1994

Mediation of most atypical effects by species homologues of the beta 3-adrenoceptor.

1. A wide panel of compounds acting on beta-adrenoceptors active either in mammalian heart or in rodent digestive tract and adipose tissues, were investigated for their effects on Chinese hamster ovary cells transfected with the human or murine beta 3-adrenoceptor gene. 2. The beta 3-agonists, bucindolol, CGP 12177A and pindolol exhibited the highest binding affinities; BRL 37344, LY 79771, ICI 201651 and SR 58611A presented high potencies in stimulating adenylyl cyclase; bupranolol appeared as the most efficient beta 3-antagonist. 3. This pharmacological analysis further established that the beta 3-adrenoceptor is the prototype of the adipose tissue atypical beta-adrenoceptor, since these receptors share a number of pharmacological properties which differ strikingly from those of beta 1- and beta 2-adrenoceptors: low affinities for conventional beta-adrenoceptor agonists and antagonists, high potencies for novel compounds active in adipose tissues, partial agonistic activities for several beta 1/beta 2-antagonists. 4. Although the pharmacological profiles of the human and murine beta 3-receptor were very similar, some quantitative or even qualitative differences were observed for particular compounds such as propranolol, which exhibited weak and partial agonistic effects at the human beta 3-receptors and antagonistic effects at the murine beta 3-receptors. These differences may result from key amino-acid substitutions between the human and the murine beta 3-receptor sequences, which may alter the binding site or signal processing. 5. Compounds active on atypical beta-sites of other tissues such as heart and digestive tract were also potent on the beta 3-adrenoceptor expressed in Chinese hamster ovary cells, suggesting that this receptor mediates most of the atypical properties described in various tissues, and that differences in ligand effects may result from tissue-related specificities.

Adenylyl Cyclases↗
PubMed1994

PCR expression analysis of the estrogen-inducible gene BCEI in gastrointestinal and other human tumors.

A polymerase chain reaction (PCR) assay was developed to test for tumor cell specific expression of the BCEI gene. This new marker gene, reported at first for human breast cancer, was found specifically active in various gastrointestinal carcinomas by previously applying immunohistochemistry and RNA (Northern blot) analysis. Presently, by using reverse transcription-PCR analysis, a series of primary tumor tissues and established tumor cell lines were tested for BCEI transcription. This approach was compared to immunostaining achieved by an antibody directed against the BCEI gene's product. The result demonstrate the superior sensitivity of PCR by indicating the gene's expression in cases where immunohistochemical testing remained negative.

Base Sequence↗
PubMed1994

Sequence patterns and hybridization analysis of clones generated by Alu-PCR.

A series of clones from an Alu-PCR library were analysed in more detail. Characterization by Southern blot hybridization and sequencing displayed several features common to all probes generated by this approach: Short length of the PCR-products as well as the presence of homologous regions on both ends resulted in a limited feasibility for filter hybridization and a low probability of restriction length polymorphisms. In addition, a series of different short repeats at the 3'-ends of Alu-repeats and present in the generated probes offers a rich source of potential variable sites accessible by PCR.

Animals↗
PubMed1993

Cosmid mapping and locus linkage within the human chromosomal region 22q13.1.

Many human meningiomas show loss of heterozygosity at distal loci but retain constitutional heterozygosity at one or more proximal loci of 22q. Molecular analysis indicted deletions involving at least the region 22q12.3-qter. In this region, distal to myoglobin, the putative meningioma locus ought to be expected. Long-range mapping was performed around two loci from 22q12.3-q13.1 (D22S16 and PDGFB, the most proximal locus to be lost in meningioma). D22S16, originally assigned to 22q13-qter by isotopic in situ hybridization, was placed in the vicinity of PDGFB by utilizing a set of somatic cell hybrids, an assignment confirmed by fluorescence in situ hybridization (FISH) of a cosmid clone containing the D22S16 locus. Moreover, pulsed field gel electrophoresis suggests a close linkage of both markers within 630 kb.

Arachnoid↗
PubMed1993

Retrospective analysis of prognostic significance of the estrogen-inducible pS2 gene in male breast carcinoma.

BACKGROUND: The estrogen-inducible pS2 gene, originally isolated from a breast cancer cell line, is correlated with hormone-dependent female breast tumors and its expression is associated with longer overall and disease-free survival. METHODS: The authors have investigated 38 samples of carcinomas of the male breast for pS2 expression by using a monoclonal antibody. The immunostaining was compared with clinical data, in particular, to the progesterone receptor status, to assess a possible prognostic value of this parameter. RESULTS: Although most cases (27 of 38) were immunopositive (i.e., above the 5th percentile of immunoreactive cells), no correlation with tumor grade and survival was notable. CONCLUSIONS: Therefore, on the contrary to the situation in female breast cancer, pS2 activity failed to constitute a new prognostic parameter in male breast carcinomas.

Adult↗
PubMed1993

Telomeric associations and loss of telomeric DNA repeats in renal tumors.

In a series of cytogenetically analyzed renal tumors of different histological types (chromophobe carcinoma, clear-cell carcinoma, chromophilic carcinoma, and oncocytoma) and normal renal tissue, the length of the telomeric DNA repeats was assessed using a (TTAGGG)3 oligonucleotide probe. A strong correlation was noted between pronounced telomere shortening and the appearance of telomeric associations of chromosomes. These data suggest an etiologic role of the loss of telomeric DNA repeats in the formation of telomeric associations and a possible involvement of this mechanism in the pathogenesis of chromosome aberrations in human tumors.

Adenocarcinoma↗
PubMed1993

Application of DNA filter hybridization and PCR to distinguish between human and non-human tissues of poor quality.

The present report describes a novel approach for the identification of human or non-human specimens after long-term storage in a badly preserved state. The application of the PCR-technique (polymerase chain reaction) using human-specific primers as well as Southern blot (filter) hybridization of the sample DNA to a primate-specific DNA probe enabled us to extend the positive identification beyond the limits of conventional methods such as serological or morphological examinations.

Animals↗