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Biomedical subjects

A M Spiegel

Publications and source records attributed to A M Spiegel.

At least 19 recordsLinked to original sources
PubMed1992

Allelic loss from chromosome 11 in parathyroid tumors.

Parathyroid tumors may occur in a sporadic fashion or, more rarely, as part of a familial syndrome (such as familial multiple endocrine neoplasia type I). The MENI gene has been mapped by linkage analysis to chromosome 11 at band q11-q13, and presumably acts as a tumor suppressor gene. In the present study, which is an extension of our previous studies, we examined 41 parathyroid tumors from patients with familial multiple endocrine neoplasia type I and 61 sporadic parathyroid tumors with markers on chromosome 11, to assess the extent of allelic loss in those tumors. Twenty-four of the MENI-associated tumors (58%) and 16 of the sporadic parathyroid tumors (26%) displayed allelic loss from chromosome 11. The region of overlap of the allelic losses in the MENI-associated tumors enables us to place the MENI gene between PGA centromerically and INT2 telomerically, a region spanning about 7.5 cM. Taken together with locus ordering by linkage analysis, this clearly localizes the MENI gene telomeric to the PGA locus. Our inability to detect allelic loss on chromosome 11 in some parathyroid tumors suggests the existence of other genes involved in the development and/or progression of this subgroup of presumably monoclonal tumors; or that localized events involving the 11q tumor suppressor gene have occurred in some parathyroid tumors whose detection is beyond the sensitivity of our analysis; or that at least some of the specimens analyzed were in fact primarily hyperplastic parathyroid tissue.

Adenoma↗
PubMed1992

High level expression of transfected G protein alpha i3 subunit is required for plasma membrane targeting and adenylyl cyclase inhibition in NIH 3T3 fibroblasts.

The alpha subunits of pertussis toxin-sensitive G proteins Gi1, Gi2 and Gi3 have been shown to inhibit adenylyl cyclase in transfected cells. However, Gi3 has recently been associated with protein transport and localized to the Golgi apparatus in a number of cell lines, rather than to the plasma membrane. We studied NIH 3T3 clones stably expressing different levels of a constitutively activated mutant of the alpha subunit of Gi3 (alpha i3-Q204L). Transfected alpha i3 subunits were localized to the Golgi apparatus in all NIH 3T3 clones. In clones expressing alpha i3-Q204L at high levels, alpha i3 subunits were also localized to the plasma membrane. Those clones which demonstrated expression of alpha i3 at the plasma membrane showed a 40% to 60% inhibition of forskolin-induced cAMP accumulation. Transfected NIH 3T3 clones in which plasma membrane alpha i3 was undetectable, did not show inhibition of forskolin-induced cAMP accumulation. These data suggest that, unless high expression is achieved in transfected cells, alpha i3 is targeted predominantly to the Golgi, not to the plasma membrane, and does not control adenylyl cyclase activity in NIH 3T3 fibroblasts.

3T3 Cells↗
PubMed1992

Differential isoprenylation of carboxy-terminal mutants of an inhibitory G-protein alpha-subunit: neither farnesylation nor geranylgeranylation is sufficient for membrane attachment.

To determine the effect of protein isoprenylation with farnesyl vs geranylgeranyl groups on membrane association in vivo, COS cells were transfected with cDNAs encoding the wild-type G-protein alpha i1 (WT) subunit, the soluble nonmyristoylated G-protein alpha i1 glycine to alanine mutant (GA), a double mutant in which the carboxy-terminal residues CGLF of GA were mutated to CVLS (GA-CVLS), and a double mutant in which the carboxy terminus of GA was mutated to CALL (GA-CALL). As opposed to the WT and GA proteins, the GA-CVLS and GA-CALL proteins were not pertussis toxin substrates nor were they recognized by antibodies that recognize the nonmutated alpha i1 carboxy terminus. Only the GA-CVLS and GA-CALL proteins incorporated [3H]mevalonate in the form of a farnesyl and a geranylgeranyl moiety, respectively. Subcellular localization, as assessed by immunoblotting and immunoprecipitation, revealed that the WT protein localizes almost exclusively to the membrane fraction, whereas the GA, GA-CVLS, and GA-CALL proteins localize predominantly to the soluble fraction. The soluble GA-CVLS and GA-CALL proteins were not carboxyl methylated, but the small amount localized to the membrane was partially carboxyl methylated. These results indicate that neither farnesylation nor geranylgeranylation is sufficient alone to lead to membrane association.

Amino Acid Sequence↗
PubMed1992

Inhibition of thyrotropin-induced DNA synthesis in thyroid follicular cells by microinjection of an antibody to the stimulatory G protein of adenylate cyclase, Gs.

Thyrotropin (TSH) is an important regulator of thyroid follicular cells. While its role in the maintenance of differentiated functions is undisputed, its role as a mitogen is less clear. TSH induces DNA synthesis and cell proliferation in some cells, while in others, TSH is mitogenic only in the presence of additional growth factors such as insulin-like growth factor-1. TSH causes elevations in intracellular cAMP and is thought to utilize this second messenger system in its mitogenic action. We studied TSH as a mitogen in Wistar rat thyroid cells (WRT) (Brandi, M. L., Rotella, C. M., Mavilia, C., Franceschelli, F., Tanini, A., and Toccafondi, R. (1987) Mol. Cell. Endocrinol. 54, 91-103) and examined the role of the guanine nucleotide binding protein, Gs, in its mitogenic action. WRT cells synthesized DNA in response to TSH and elevations in cAMP. In addition, TSH caused a rapid stimulation of an indicator gene whose expression is regulated by cAMP response elements. Following microinjection of an inhibitory polyclonal antibody raised against the Gs protein, both TSH-induced changes in gene expression and DNA synthesis were significantly reduced. These results demonstrate that virtually all of the mitogenic action of TSH is transduced through the Gs protein in WRT cells, presumably through the regulation of adenylate cyclase. Whether all or only part of TSH action is mediated by cAMP and the cAMP-dependent protein kinase remains to be determined.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Site-specific antibodies directed against G protein beta and gamma subunits: effects on alpha and beta gamma subunit interaction.

Little is known about the specific domains of G protein beta and gamma subunits which interact with each other and with the alpha subunit. We used site-specific anti-peptide antibodies directed against beta and gamma subunits to investigate domains on beta and gamma subunits involved in alpha subunit interaction. Antibodies included four against the transducin (Gt) beta subunit (residues 1-10 = MS, 127-136 = KT, 256-265 = RA, and 330-340 = SW) and two against the gamma subunit (residues 2-12 = PV and 58-68 = PE). All antisera, when affinity-purified on peptide columns, yielded antibodies capable of recognizing the denatured cognate subunit on immunoblots, but only RA, SW, PV, and PE recognized native beta gamma t subunits. Affinity purification of MS and KT antisera on columns of immobilized native Gt yielded antibodies capable of recognizing native beta gamma t subunits. The functional effects of each antibody preparation on alpha t-beta gamma t interaction were assessed by assaying the ability of the preparations to immunoprecipitate beta gamma t subunits in the presence of excess alpha subunits and by testing the inhibition of beta gamma t-dependent ADP-ribosylation of alpha t-subunits catalyzed by pertussis toxin. On the basis of the results, we conclude that the domains on beta gamma t which may be directly involved in alpha t-beta gamma t interaction include the extreme amino terminus, residues 127-136 and 256-265 of beta t, and the carboxyl terminus of gamma t.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1992

Inhibition of DNA synthesis in living cells by microinjection of Gi2 antibodies.

Heterotrimeric guanine nucleotide binding proteins function in the coupling of a diverse span of cell surface receptors to a variety of intracellular signaling pathways, some of which stimulate cellular proliferation. With the recent discovery that mutated forms of G proteins are present in specific tumors, there has been an increased interest in the determination of the role of specific subtypes of G proteins in the regulation of cellular growth. We have attempted to determine which subtypes of G proteins are directly involved in serum-stimulated DNA synthesis through microinjection of inhibitory antibodies into living cells. Inhibitory rabbit polyclonal antibodies directed against specific Gi alpha subunits were introduced into living Balb/c 3T3 fibroblasts by microinjection, and the effect upon serum-stimulated DNA synthesis was examined. Results of these experiments indicate that Gi2 plays a direct role in serum-stimulated DNA synthesis in living cells and suggest that G proteins may function in a variety of mitogenic signaling pathways initiated by serum growth factors.

3T3 Cells↗
PubMed1992

A heterozygous 4-bp deletion mutation in the Gs alpha gene (GNAS1) in a patient with Albright hereditary osteodystrophy.

Several heterozygous mutations within the gene encoding the alpha-subunit of Gs (GNAS1), the G protein that stimulates adenylyl cyclase, have been previously identified in patients with Albright hereditary osteodystrophy (AHO). We have now identified a fourth GNAS1 mutation from an AHO patient. Amplification by the polymerase chain reaction (PCR) of a genomic fragment encompassing GNAS1 exons 7 and 8 from one patient resulted in a product with aberrant migration on nondenaturing polyacrylamide and agarose gels. Direct DNA sequencing identified a 4-bp deletion in one allele of exon 7 encoding a frameshift with a premature stop codon. Analysis of lymphocyte RNA by reverse transcription-PCR and direct sequencing showed that the GNAS1 allele bearing the mutation is not expressed as mRNA. Consistent with this, Northern analysis revealed an approximate 50% deficiency in steady-state levels of GNAS1 mRNA. These findings further illustrate the heterogeneity of GNAS1 gene defects in AHO.

Amino Acid Sequence↗
PubMed1992

G proteins in cellular control.

In the past year, cDNA cloning has revealed substantial diversity in G protein alpha, beta and gamma subunits. The number of cellular functions recognized to be controlled by G proteins is also increasing. Most G proteins are associated with the cytoplasmic surface of the plasma membrane, and molecular mechanisms for membrane association of certain G protein subunits have been defined recently. Mutations in G protein subunits, both artificially induced and naturally occurring, have provided important insights into G protein structure and function.

Animals↗
PubMed1992

Amino- and carboxy-terminal deletion mutants of Gs alpha are localized to the particulate fraction of transfected COS cells.

To elucidate the structural basis for membrane attachment of the alpha subunit of the stimulatory G protein (Gs alpha), mutant Gs alpha cDNAs with deletions of amino acid residues in the amino and/or carboxy termini were transiently expressed in COS-7 cells. The particulate and soluble fractions prepared from these cells were analyzed by immunoblot using peptide specific antibodies to monitor distribution of the expressed proteins. Transfection of mutant forms of Gs alpha with either 26 amino terminal residues deleted (delta 3-28) or with 59 amino terminal residues deleted (delta 1-59) resulted in immunoreactive proteins which localized primarily to the particulate fraction. Similarly, mutants with 10 (delta 385-394), 32 (delta 353-384), or 42 (delta 353-394) amino acid residues deleted from the carboxy terminus also localized to the particulate fraction, as did a mutant form of Gs alpha lacking amino acid residues at both the amino and carboxy termini (delta 3-28)/(delta 353-384). Mutant and wild type forms of Gs alpha demonstrated a similar degree of tightness in their binding to membranes as demonstrated by treatment with 2.5 M NaCl or 6 M urea, but some mutant forms were relatively resistant compared with wild type Gs alpha to solubilization by 15 mM NaOH or 1% sodium cholate. We conclude that: (a) deletion of significant portions of the amino and/or carboxyl terminus of Gs alpha is still compatible with protein expression; (b) deletion of these regions is insufficient to cause cytosolic localization of the expressed protein. The basis of Gs alpha membrane targeting remains to be elucidated.

Amino Acid Sequence↗
PubMed1992

Results of a multidisciplinary strategy for management of mediastinal parathyroid adenoma as a cause of persistent primary hyperparathyroidism.

Persistent primary hyperparathyroidism due to mediastinal parathyroid adenoma was effectively treated by either angiographic ablation or median sternotomy in this study of 49 patients managed at the National Institutes of Health since 1977. Each patient presented here with symptomatic persistent primary hyperparathyroidism after failed initial surgical procedures done at other institutions. Each patient underwent extensive parathyroid localization procedures, including selective angiography, and most had a parathyroid adenoma localized to the mediastinum. Angiographic ablation, the deliberate injection of large doses of contrast material into the artery that selectively perfuses the adenoma, was initially successful in 22 of 30 procedures (73%) in 27 patients. Long-term control of persistent primary hyperparathyroidism was achieved in 17 of 27 patients (63%) by angiographic ablation. Each unsuccessful ablation could be easily salvaged by surgical resection. Surgical resection of the parathyroid adenoma by median sternotomy achieved immediate success in 24 of 24 procedures (p2 less than 0.02 versus ablation), and long-term cure in 23 of 23 evaluable patients (p2 less than 0.001 versus ablation). However, ablation did have benefits for the patients in whom it was successfully performed. It was associated with a significantly shorter hospital stay (median, 6 days versus 9 days for sternotomy, p2 less than 0.003), much less pain, and easier recuperation. Complications of each procedure were transient and similar in both groups. Operative resection is the most effective single means to eradicate mediastinal parathyroid adenoma; however, angiographic ablation can provide similar long-term control of hyperparathyroidism in 63% of patients with less pain and shorter convalescence than that seen in patients after median sternotomy. Our results suggest that angiographic ablation should be attempted as the initial procedure for patients with persistent primary hyperparathyroidism caused by an angiographically identified mediastinal parathyroid adenoma. Operation can be reserved for those who fail ablation.

Adenoma↗
PubMed1992

Was it murder or insanity? Reactions to a successful paroxysmal insanity plea in 1865.

This 1865 Harris/Burroughs trial marked the first time that the paroxysmal [temporary] insanity plea was supported by expert medical testimony in a U.S. courtroom. Jurors agreed that the female defendant was insane at the time of the shooting due to being "crossed in love and suffering from painful dysmenorrhea." Attitudinal stances of the legal system, health professionals, newspapers and the public are illustrated. By merely changing the antebellum language, the reactions could be recycled into our current tabloid newspapers and titillating TV programs as if the tragedy occurred yesterday.

Adolescent↗
PubMed1991

Activating mutations of the stimulatory G protein in the McCune-Albright syndrome.

BACKGROUND: The McCune-Albright syndrome is a sporadic disease characterized by polyostotic fibrous dysplasia, café au lait spots, sexual precocity, and hyperfunction of multiple endocrine glands. These manifestations may be explained by a somatic mutation in affected tissues that results in activation of the signal-transduction pathway generating cyclic AMP (cAMP). We analyzed DNA from tissues of patients with the McCune-Albright syndrome for the presence of activating mutations of the gene for the alpha subunit of the G protein (Gs alpha) that stimulates cAMP formation. METHODS: Genomic DNA fragments encompassing regions (exons 8 and 9) previously found to contain activating missense mutations of the Gs alpha gene (gsp mutations) in sporadically occurring pituitary tumors were amplified in tissues from four patients with the McCune-Albright syndrome by the polymerase chain reaction. The amplified DNA was analyzed for mutations by denaturing gradient gel electrophoresis and allele-specific oligonucleotide hybridization. RESULTS: We detected one of two activating mutations within exon 8 of the Gs alpha gene in tissues from all four patients, including affected endocrine organs (gonads, adrenal glands, thyroid, and pituitary) and tissues not classically involved in the McCune-Albright syndrome. In two of the patients, histidine was substituted for arginine at position 201 of Gs alpha, and in the other two patients cysteine was substituted for the same arginine residue. In each patient the proportion of cells affected varied from tissue to tissue. In two endocrine organs, the highest proportion of mutant alleles was found in regions of abnormal cell proliferation. CONCLUSIONS: Mutations within exon 8 of the Gs alpha gene that result in increased activity of the Gs protein and increased cAMP formation are present in various tissues of patients with the McCune-Albright syndrome. Somatic mutation of this gene early in embryogenesis could result in the mosaic population of normal and mutant-bearing tissues that may underlie the clinical manifestations of this disease.

Adolescent↗
PubMed1991

Activating and inactivating mutations of the alpha subunit of Gi2 protein have opposite effects on proliferation of NIH 3T3 cells.

Previous studies have demonstrated that mutations of highly conserved residues in the alpha subunit of Gs (alpha s) can inhibit either the intrinsic GTPase activity (glutamine-227 to leucine, Q227L) or the ability of the protein to be activated by GTP (glycine-226 to alanine, G226A). We stably transfected NIH 3T3 cells with cDNAs encoding Gi2 alpha subunit (alpha i2) containing either wild-type sequence or the homologous mutations Q205L and G204A. High expression of wild-type alpha i2, Q205L alpha i2, and G204A alpha i2 was confirmed in transfected cells by immunoblot analysis. The overexpression of all three alpha i2 proteins was accompanied by an increase in beta-subunit expression. Q205L alpha i2 was a poor substrate for ADP-ribosylation by pertussis toxin as compared with wild-type alpha i2. Expression of Q205L alpha i2 markedly decreased forskolin- or cholera toxin-stimulated intracellular cAMP levels in intact cells, confirming the constitutively activated state of the protein. In contrast, G204A alpha i2 increased intracellular cAMP and was resistant to guanosine 5'-[gamma-thio]triphosphate-induced inhibition of ADP-ribosylation by pertussis toxin, as expected for an inactive alpha i2. Transfection of wild-type, Q205L, or G204A alpha i2 cDNA did not induce focus formation of NIH 3T3 cells. However, overexpression of Q205L alpha i2 induced a decreased serum requirement, a reduced doubling time, and an 8- to 10-fold increase in [3H]thymidine incorporation. Q205L alpha i2 cells formed small colonies in soft agar, demonstrating some degree of anchorage-independent proliferation. Expression of G204A alpha i2 slowed the growth of NIH 3T3 cells. We conclude that alpha i2 plays an important role in regulation of fibroblast growth.

3T3 Cells↗