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Biomedical subjects

N Blin

Publications and source records attributed to N Blin.

At least 91 records · Page 5Linked to original sources
PubMed1993

Expression of the breast cancer-associated protein pS2 in adenosquamous carcinomas of the gastrointestinal tract.

The breast cancer-associated protein pS2 is also present in many human gastrointestinal tumors. In contrast to breast carcinomas, gastrointestinal tumors do not express estrogen receptors, indicating that the expression of pS2 is not estrogen-dependent. The pS2 expression was analyzed in 14 adenosquamous tumors of the human gastrointestinal tract. The aim was to investigate if the cell type specific localization of pS2 was limited to the glandular part. The data clearly confirm such a specific compartmentation of the pS2 expression, suggesting pS2 to be a secreted protein. Due to the specific expression, pS2 may become a new and useful diagnostic marker of adenocarcinoma.

Adenocarcinoma↗
PubMed1993

High level functional expression of human beta 1-adrenergic receptor in baculovirus-infected cells screened by a rapid in situ procedure.

A novel screening assay for the identification of baculovirus infected cells expressing membrane receptors was developed by using a replica transfer technique. Sf9 cells were cotransfected with wild type baculoviral DNA and the transfer vector pVL941-beta 1 containing the coding region of the human beta 1-adrenergic receptor gene. Infected cells embedded in agarose were incubated with [125I]-iodocyanopindolol and transferred onto filters that were subsequently autoradiographed. This procedure resulted in the isolation of recombinant baculoviruses that expressed beta 1-adrenergic receptors. Binding assays carried out with [125I]-ICYP indicated that more than 600,000 receptors were expressed per cell, the highest level noted so far for this receptor in genetically engineered cells. Sf9 cells expressing the beta 1-AR were analysed by ligand binding, competition experiments, adenylyl cyclase stimulation and photoaffinity labeling. These cells express a homogenous population of receptors and display the known pharmacological properties of beta 1-AR in human tissues.

Adenylyl Cyclases↗
PubMed1993

Structural and conformational features determining selective signal transduction in the beta 3-adrenergic receptor.

With respect to the beta 1- and beta 2-adrenergic receptors (ARs), the beta 3-AR induces specific physiological effects in a few target tissues and exhibits atypical pharmacological properties that distinguish it unambiguously from its counterparts. Therefore, the beta 3-AR represents a suitable model to study the molecular mechanism responsible for receptor subtype selectivity and specificity. Potent beta 3-AR ligands newly characterized in Chinese hamster ovary cells expressing the beta 3-AR were also evaluated in Chinese hamster ovary cells expressing beta 1- and beta 2-ARs and were classified into three groups according to their pharmacological properties. Among the beta 1/beta 2/beta 3 agonists BRL 37344 and LY 79771 exhibit beta 3 selectivity in stimulating adenylyl cyclase; among the beta 1/beta 2 antagonists displaying beta 3 agonistic effects ICI 201651 exhibits beta 3-AR binding selectivity, whereas among the beta 1/beta 2/beta 3 antagonist class bupranolol is the most efficient (but not selective) beta 3-AR antagonist. The structures of these ligands were simulated and compared using computer-generated molecular modeling. Structure-activity relationship analysis indicates that potent or selective beta 3-AR compounds, in addition to possessing a pharmacophore common to all beta-AR ligands, contain a long and bulky alkylamine substituent moiety, which is able to adopt and exchange extended and stacked conformations. Computerized three-dimensional models of the beta 1-, beta 2-, and beta 3-AR binding sites show that more bulky amino acid side chains point inside the groove of the beta 1 and beta 2 sites, compared with the beta 3 site, in a region implicated in signal processing. The long alkylamine chain of compounds behaving as beta 1/beta 2 antagonists and beta 3 agonists may thus adopt either a stacked conformation in the encumbered beta 1- and beta 2-AR sites, leading to antagonistic effects, or an extended conformation in the less encumbered beta 3 site, thus interacting with specific residues implicated in signal transduction.

Adrenergic beta-Agonists↗
PubMed1992

In vitro study of a novel atypical beta-adrenoceptor agonist, SM-11044.

SM-11044 (L-threo-3-(3,4-dihydroxyphenyl)-N-[3-(4-fluorophenyl) propyl] serine pyrrolidine amide hydrobromide) stimulated the relaxation of guinea pig ileum (EC50: 3.0 x 10(-8) M), trachea (EC50: 1.3 x 10(-7) M), lung parenchyma (EC50: 2.1 x 10(-6) M) and the increase in right atrial rate (EC50: 6.9 x 10(-6) M). It also induced lipolysis of rat white adipocytes (EC50: 1.2 x 10(-6) M). Both the relaxant response of ileum and the lipolytic response of adipocytes to SM-11044 were resistant to inhibition by propranolol (10(-6) M), but were antagonized by cyanopindolol (10(-6) M). In contrast, the responses to SM-11044 in trachea, lung parenchyma and right atrium were almost completely abolished by propranolol (10(-6) M). Furthermore, the selectivity of SM-11044 relative to isoproterenol was ileum greater than adipocytes greater than trachea (beta 2) greater than lung (beta 2) greater than atrium (beta 1). These results suggest that SM-11044 is a selective agonist of atypical beta-adrenoceptors that are resistant to antagonism by propranolol but sensitive to cyanopindolol. The receptor binding and adenylate cyclase stimulating activity of SM-11044 were also examined on transfected Chinese hamster ovary cells expressing human beta 1-, beta 2- or beta 3-adrenoceptors. SM-11044 inhibited the binding of [125I]iodocyanopindolol to the three types of receptors in a concentration-dependent manner. The selectivity in terms of Ki values was beta 3 (Ki: 1.3 x 10(-6) M) greater than beta 2 (Ki: 4.1 x 10(-6) M) greater than beta 1 (Ki: 18.1 x 10(-6) M)-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1992

DNA-protein interaction analysis using polyacrylamide gel electrophoresis and a simple and sensitive UV crosslinking procedure.

A simple and reproducible technique for DNA-protein interaction analysis is described using UV crosslinking and polyacrylamide gel electrophoresis, leading to visualization of the complexes as distinct and strong signals. It avoids incorporation of bromodeoxyuridine (BrdU) into DNA and requires no special equipment. It was successfully applied to an oligonucleotide sequence from within the first intron of the mouse myb proto-oncogene and nuclear extracts from a myb-expressing cell line.

Base Sequence↗
PubMed1992

A two-colour technique for chromosome in situ hybridization in tissue sections.

By extending non-isotopic in situ hybridization of DNA probes (targeted to metaphase chromosomes or interphase nuclei) to hybridization using tissue sections, additional topological information on the DNA structure and specific alterations can be obtained. We have established a method for the application of two different, chromosome-specific probes labelled with two colour dyes allowing simultaneous detection of two-colour signals. This method was tested in and is applicable to tissue sections of various origins. To demonstrate its sensitivity, prostate carcinomas (either as cryosections or as sections from paraffin blocks) were investigated for the presence or absence of chromosomes 1 and Y. The technique presented here, comparable to immunohistochemical staining, is particularly useful for routine application in diagnostic laboratories and testing of fresh or archival material.

Carcinoma↗
PubMed1992

A non-alphoid repetitive DNA sequence from human chromosome 21.

A non-alphoid repetitive DNA from human chromosome 22, consisting of a 48-bp motif, shows homology to both G-group chromosomes in the gorilla, thus indicating the presence of additional repeat family members on further human chromosomes. Therefore, we screened a chromosome-21-specific cosmid library using this repetitive sequence from chromosome 22 (D22Z3). Some 40-50 cosmid clones were positive in tests for hybridization. One of the clones giving the strongest signals was digested with EcoRI/PstI, which we knew to cut frequently within the repeats; this resulted in fragments containing repeat units only. The fragments were subcloned into plasmid vector pTZ 19. Sequence-analysis of a 500-bp insert showed ten copies of a 48-bp repeat similar to D22Z3, with about 15% sequence deviation from the chromosome 22 consensus sequence. In situ hybridization of the newly isolated recombinant established its chromosome 21 specificity at high stringency. Physical mapping by pulsed field gel electrophoresis placed this new repeat in close vicinity to the chromosome 21 alphoid repeat. No cross-hybridization with other mammalian genomes except for those of apes was observed. The locus has been designated D21Z2 by the Genome Data Base. A gel mobility shift assay indicated that this repetitive motif has protein-binding properties.

Animals↗
PubMed1992

Search for putative suppressor genes in meningioma: significance of chromosome 22.

Cytogenetic and molecular studies of various solid tumors have indicated that a series of different chromosomal regions may be deleted in the tumor genome. Usually, losses of heterozygosity are observed and, from this finding, the presence of specific genes acting as tumor suppressors has been deduced. In particular tumors, however, only a single chromosome site appears to be affected. Therefore, we have carried out a study of human meningioma, investigating 7 such putative suppressor regions by applying twelve site-specific DNA markers. In 6 out of 19 tumors, we exclusively found loss of heterozygosity for markers of the long arm of chromosome 22; none of the tumors showed statistically significant additional allelic losses for the regions 1p, 3p, 5p, 5q, 11p, 13q, 17p. Our data support the long-standing observation that only losses of or within chromosome 22 are associated with the development of meningiomas. Other suppressor regions are apparently not involved.

Adult↗
PubMed1992

Assignment of the gene for human spasmolytic protein (hSP/SML1) to chromosome 21.

A human cDNA corresponding to the porcine pancreatic spasmolytic protein (PSP) was isolated, and the recombinant clone was originally termed hSP for human spasmolytic protein. Later, the term SML1 for spasmolysin was suggested for the human gene. This protein shows a remarkable sequence homology to pS2, a protein coded by an estrogen-induced gene isolated from the breast carcinoma cell line MCF-7. Although, at the DNA level, the gene sequences pS2 and hSP/SML1 display insufficient homology for cross-hybridization, their expression in tumor cells occurs with remarkable coordination. The human pS2 gene sequence has been assigned to chromosome 21, and we have therefore attempted to map the hSP/SML1 gene by using cDNA and Southern blotting of genomic DNAs from a panel of human-rodent somatic cell hybrids carrying different complements of human chromosomes. Interestingly, the hSP/SML1 gene is also localized on chromosome 21.

Blotting, Southern↗
PubMed1992

Nonradioactive southwestern analysis using chemiluminescent detection.

Replacing radioactively labeled probes by nonradioactive ones and detection by chemiluminescence instead of colorimetry allows a nonhazardous handling and offers the possibility of easily reprobing filters in Southwestern analysis. Using the described procedure, we were able to determine the molecular weight of DNA-binding proteins and achieve a high signal-to-noise ratio.

Animals↗
PubMed1992

Association of the human spasmolytic polypeptide and an estrogen-induced breast cancer protein (pS2) with human pancreatic carcinoma.

The human pS2 gene, isolated from the breast carcinoma cell line MCF-7 and shown to be under estrogen transcriptional control in a subclass of breast cancer cells was reported to be secreted in normal stomach surface epithelial cells, whereas additional gastrointestinal tissues like pancreas and colon do not secrete pS2 at all. In porcine pancreas, a spasmolytic polypeptide (sharing domains of homology with pS2) was observed; a corresponding human gene (hSP) was shown to be active in normal stomach mucosa. hSP and pS2 gene activity in normal and neoplastic pancreas tissues was then compared. Whereas both genes are inactive in normal pancreatic cells, activation of the pS2 sequence in a primary pancreatic carcinoma cell culture and in 23 tumor tissues was noted when investigated by immunostaining. In all cases when pS2 showed a regular 0.6 kb transcript, hSP displayed a transcript of 0.7 kb. Six of these tumors showed a reduced pS2 immunoreactivity and, at the same time, aberrant pS2 mRNA bands and a complete shut-down of the hSP gene were noted. In one case, whereas normal pancreas remained negative, the corresponding tumor and its metastasis displayed regular transcripts of pS2 and hSP. This remarkably high correlation suggests that pS2 and hSP expression in the pancreatic tumors, but not in their corresponding healthy tissue is significantly linked to molecular steps leading to tumorigenesis.

Adenocarcinoma↗
PubMed1991

Atypical beta-adrenergic receptor in 3T3-F442A adipocytes. Pharmacological and molecular relationship with the human beta 3-adrenergic receptor.

Expression of ligand binding properties for an atypical beta-adrenergic receptor (beta-AR) subtype was studied during the adipose differentiation of murine 3T3-F442A cells and compared with that of the human beta 3-AR expressed in Chinese hamster ovary cells stably transfected with the human beta 3-AR gene (CHO-beta 3 cells) Emorine, L. J., Marullo, S., Briend-Sutren, M. M., Patey, G., Tate, K., Delavier-Klutchko, C., and Strosberg, A. D. (1989) Science 245, 1118-1121). 3T3-F442A adipocytes exhibited high and low affinity binding sites for (-)-4-(3-t-butylamino-2-hydroxypropoxy) [5,7-3H]benzimidazole-2-one ((-)-[3H]CGP-12177) (KD = 1.2 and 38.3 nM) and (-)-[125I]iodocyanopindolol ([125I]CYP) (KD = 47 and 1,510 pM). The high affinity sites corresponded to the classical beta 1- and beta 2-AR subtypes whereas the KD values of the low affinity sites for the radioligands were similar to those measured in CHO-beta 3 cells (KD = 28 nM and 1,890 pM for (-)-[3H]CGP12177 and [125I]CYP, respectively). These low affinity sites were undetectable in preadipocytes but represented about 90% of total beta-ARs in adipocytes. The atypical beta-AR and the human beta 3-AR add similarly low affinities (Ki = 3-5 microM) for (+/-)-(2-(3-carbamoyl-4-hydroxyphenoxy)ethylamino-3)-(4-(1-methyl- 4- trifluormethyl-2-imidazolyl)-phenoxy)-2-propanol methane sulfonate (CGP20712A) or erythro-(+/-)-1-(7-methylindan-4-yloxy)-3-isopropylaminob utan-2-ol (ICI118551), highly selective beta 1- and beta 2-AR antagonists, respectively, in agreement with the poor inhibitory effect of the compounds on (-)-isoproterenol (IPR)-stimulated adenylate cyclase activity. Atypical beta-AR and beta 3-AR had an affinity about 10-50 times higher for sodium-4-(2-[2-hydroxy-2-(3-chlorophenyl)ethylamino]propyl)phenoxyace tate sesquihydrate (BRL37344) than the beta 1-AR subtype. This correlates with the potent lipolytic effect of BRL37344 in adipocytes. The rank order of potency of agonists in functional and binding studies was BRL37344 greater than IPR less than (-)-norepinephrine greater than (-)-epinephrine both in 3T3 adipocytes and CHO-beta 3 cells. As in CHO-beta 3 cells, the classical beta 1- and beta 2-antagonists CGP12177, oxprenolol, and pindolol were partial agonists in adipocytes. Although undetectable in preadipocytes, a major mRNA species of 2.3 kilobases (kb) and a minor one of 2.8 kb were observed in adipocytes by hybridization to a human beta 3-AR specific probe.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗
PubMed1991

Complete nucleotide sequence of the gene for human heparin cofactor II and mapping to chromosomal band 22q11.

Heparin cofactor II (HCII) is a 66-kDa plasma glycoprotein that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. Clones comprising the entire HCII gene were isolated from a human leukocyte genomic library in EMBL-3 lambda phage. The sequence of the gene was determined on both strands of DNA (15,849 bp) and included 1749 bp of 5'-flanking sequence, five exons, four introns, and 476 bp of DNA 3' to the polyadenylation site. Ten complete and one partial Alu repeats were identified in the introns and 5'-flanking region. The HCII gene was regionally mapped on chromosome 22 using rodent-human somatic cell hybrids, carrying only parts of human chromosome 22, and the chronic myelogenous leukemia cell line K562. With the cDNA probe HCII7.2, containing the entire coding region of the gene, the HCII gene was shown to be amplified 10-20-fold in K562 cells by Southern analysis and in situ hybridization. From these data, we concluded that the HCII gene is localized on the chromosomal band 22q11 proximal to the breakpoint cluster region (BCR). Analysis by pulsed-field gel electrophoresis indicated that the amplified HCII gene in K562 cells maps at least 2 Mbp proximal to BCR-1. Furthermore, the HCII7.2 cDNA probe detected two frequent restriction fragment length polymorphisms with the restriction enzymes BamHI and HindIII.

Amino Acid Sequence↗
PubMed1991

Molecular characterization of the mouse beta 3-adrenergic receptor: relationship with the atypical receptor of adipocytes.

The gene encoding the murine beta 3-adrenergic receptor (beta 3AR) has been isolated. It translates into a polypeptide of 388 amino acid residues which shows 82% overall homology with the human beta 3AR. In Southern blot experiments, a probe derived from the murine beta 3AR gene hybridizes to a unique restriction fragment in the murine and human genomes. In both species, the beta 3AR gene is located on chromosome 8, in regions (8A2----8A4 in mouse, and 8p11----8p12 in man) which are conserved between mouse and man. The pharmacological profile of the mouse beta 3AR strongly resembles that of the human beta 3AR. It is characterized by a low affinity toward the radiolabelled beta-adrenergic antagonist [125I]Iodocyanopindolol and a low efficiency of other antagonists such as propranolol, ICI 118551 or CGP 20712A to inhibit cAMP production induced by isoproterenol. Another salient feature shared by the murine and the human beta 3ARs is the very potent effect of the lipolytic compound BRL 37344 on cAMP accumulation and the partial agonistic effect of the beta 1- and beta 2-adrenergic antagonists CGP 12177A, oxprenolol and pindolol. These properties are very close to those ascribed to the atypical beta AR of rodent adipocytes. In addition, Northern blot analyses indicate that the beta 3AR gene is mainly expressed in mouse brown and white adipose tissues, suggesting that the murine beta 3AR described here is the atypical beta AR involved in the control of energy expenditure in fat tissue.

3T3 Cells↗